J Tissue Eng Regen Med - 2014 - Detsch - The Role of Osteoclasts in Bone Tissue Engineering

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE REVIEW
J Tissue Eng Regen Med 2015; 9: 1133–1149.

Published online 29 January 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1851
The role of osteoclasts in bone tissue engineering

Rainer Detsch* and Aldo R. Boccaccini
Institute of Biomaterials, University of Erlangen-Nuremberg, Germany
Abstract
The success of scaffold-based bone regeneration approaches strongly depends on the performance of
the biomaterial utilized. Within the efforts of regenerative medicine towards a restitutio ad integrum
(i.e. complete reconstruction of a diseased tissue), scaffolds should be completely degraded within an
adequate period of time. The degradation of synthetic bone substitute materials involves both chem-
ical dissolution (physicochemical degradation) and resorption (cellular degradation by osteoclasts).
Responsible for bone resorption are osteoclasts, cells of haematopoietic origin. Osteoclasts play also
a crucial role in bone remodelling, which is essential for the regeneration of bone defects. There is,
however, surprisingly limited knowledge about the detailed effects of osteoclasts on biomaterials
degradation behaviour. This review covers the relevant fundamental knowledge and progress made
in the field of osteoclast activity related to biomaterials used for bone regeneration. In vitro studies
with osteoclastic precursor cells on synthetic bone substitute materials show that there are specific
parameters that inhibit or enhance resorption. Moreover, analyses of the bone–material interface
reveal that biomaterials composition has a significant influence on their degradation in contact with
osteoclasts. Crystallinity, grain size, surface bioactivity and density of the surface seem to have a less
significant effect on osteoclastic activity. In addition, the topography of the scaffold surface can be
tailored to affect the development and spreading of osteoclast cells. The present review also
highlights possible areas on which future research is needed and which are relevant to enhance our
understanding of the complex role of osteoclasts in bone tissue engineering. Copyright © 2014 John
Wiley & Sons, Ltd.
Received 6 February 2013; Revised 18 September 2013; Accepted 20 October 2013
Keywords osteoclasts; bone tissue engineering; scaffolds; bone resorption; calcium phosphates;
bioactive glasses
1. Introduction the replacement of diseased bone by tissue-engineered

constructs is one of the key areas bound to expand in
the field of regenerative medicine (Dimitriou et al.,
Although bone tissue has a unique capacity to heal and re-
2011). In a tissue-engineering approach, target cells,
model without leaving a scar (compared to other tissues),
growth factors and biomaterial scaffolds are combined
the regeneration success is limited in the case of large de-
to form a three-dimensional (3D) graft (Kneser et al.,
fects, such as those formed after tumour removal or major
2006; Salgado et al., 2004). Finding a suitable scaffold
fractures (Petite et al., 2000). There are more than 2
for bone tissue engineering is challenging: autografts
million cases/year worldwide that require bone-grafting
(’gold standard’), allografts and xenografts are all consid-
procedures and many of these clinical situations in dental
ered, but they exhibit some issues and limitations, such as
and orthopaedic surgery need the use of suitable biomate-
risk of infection, low mechanical stability, limited
rials to enable effective bone regeneration (Laurencin
osteoconductivity, processing costs or restricted availability
et al., 2006). Numerous applications can be listed: trauma
(Zimmermann and Moghaddam, 2011; Theler, 2011;
surgery, joint-like hip and knee revision and reconstruc-
Moore et al., 2001; Bucholz, 2002). Indeed, donor site mor-
tion, spinal fusion and craniomaxillofacial surgery. Thus,
bidity, restrictions on the amount of graft material available
and concerns about the immunogenicity of autograft bone
*Correspondence to: Rainer Detsch, Institute of Biomaterials,
have prompted the search for suitable synthetic bone
University of Erlangen-Nuremberg, 91058 Erlangen, Germany. substitutes. Degradable alloplastic bone-graft materials
E-mail: rainer.detsch@ww.uni-erlangen.de used today include a wide range of biomaterials, such as
Copyright © 2014 John Wiley & Sons, Ltd.

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1134 R. Detsch and A. R. Boccaccini
polymers, metals, ceramics, bioactive glasses, cements repair, cells of the target microenvironment, such as
and composites (Rezwan et al., 2006; Giannoudis et al., inflammatory cells, fibroblasts, endothelial cells, bone
2005; Mano et al., 2007). Figure 1 summarizes the range marrow stromal cells and osteoblasts, provide the needed
of degradable bone-grafting materials which can be used growth factors. From the beginning, osteoclasts and
for scaffolds in the field of bone tissue engineering. osteoblasts are fundamentally engaged in the whole inter-
Thereby it is well known that the presence of a active process. In this context, osteocytes are also involved
macro-porous bone substitute material (scaffold) is nec- in bone remodelling; however, for biomaterials research
essary in wound and tissue repair to provide a sufficient and in tissue-engineering approaches osteocytes seem to
template to guide tissue regeneration. be less relevant than osteoblasts, fibroblasts, mesenchymal
In general, bone formation and bone resorption (or stem cells, osteoclasts and macrophages, as well as endo-
bone remodelling) are physiologically controlled by the thelial cells. For the osteoblast–osteoclast interaction,
osteoclastic resorption and synthesis of bone matrix by osteoclastic differentiation is required. Osteoclasts are
osteoblasts (Martin and Sims, 2005). To provide an over- the bone-resorbing cells and they respond to various cyto-
view of the role of osteoclast cells in these processes, the kines (Sapp, 1976; Baron et al., 1986; Chambers, 2010).
present review fills a gap in the specialized literature sum- One key factor in osteoclast development is the receptor
marizing and discussing available relevant studies in the activator of nuclear factor-κB ligand (RANKL). This signal-
field. The following sections cover relevant subjects such ling can be inhibited by osteoprotegerin (OPG), an antag-
as bone-remodelling mechanisms, development of osteoclasts onist receptor for RANKL, secreted by osteoblasts (Krast
and its characteristics, and in vitro cultivation of osteoclast- et al., 2004). Interestingly, RANKL is also a relevant
like cells. Furthermore, several parameters influencing growth factor for tissue-engineering strategies. For exam-
the resorption of biomaterials and the activity of osteo- ple, calcium phosphate cements have been coated with
clast cells in the context of bone tissue engineering are RANKL to enhance osteoclastogenesis and to improve
discussed in detail. bone remodelling (Le Nihouannen et al., 2008). Osteoclas-
tic differentiation also requires macrophage colony-stimu-
lating factor (M-CSF). The illustration in Figure 2 shows
schematically the relationship between osteoclasts and
2. Bone-healing and osteoblasts, whereby the development of bone-forming
bone-remodelling mechanism cells is induced by transforming growth factor- β (TGFβ),
interleukin-6 (IL-6) and insulin-like growth factors (IGF)
Bone regeneration is a complex process that involves the and others (Chen et al., 2004; Bruder et al., 1998; Hill
coordination of a variety of different cascades and can et al., 1995). Moreover, IL-1 is secreted by osteoclast pre-
be typically characterized by four overlapping stages cursor cells that are stimulated by an autocrine mecha-
(Einhorn, 1998); the initial inflammatory response is nism (Yao et al., 2008). Furthermore, osteoclasts secrete
followed by bone formation, vascularization and bone anabolic factors, which directly support bone formation
remodelling (Yamagiwa and Endo, 2009). During bone (Karsdal et al., 2008). One more important growth factor,
Figure 1. Classification of degradable bone substitute materials into alloplastic and biological and their subdivisions. With all these
materials several combinations for composites are possible for developing bone tissue scaffolds
Copyright © 2014 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2015; 9: 1133–1149.
DOI: 10.1002/term
Role of osteoclasts in bone tissue engineering 1135
polymeric and metallic biomaterials, which have been

found in periprosthetic tissues (Wang et al., 1997). How-
ever, osteoclasts play also a crucial role in the healthy
bone remodelling process. Osteoclasts (from the Greek
words for ’bone’ and ’broken’) were first named by Albert
Kölliker in 1873 (Kölliker, 1873; Weldon, 1898). Among
other cells, precursors of osteoclasts are present in the
bone marrow and in the peripheral blood (Wilson et al'.,
2006). These cells originate from haematopoietic precur-
sor cells and differentiate into multinucleated cells by
the cell–cell fusion of mononuclear progenitors (Chambers,
2010; Vignery, 2000; Bar-Shavit, 2007). Cell fusion is one
of the characteristic properties of osteoclasts; however,
there are several other fusing cell types, including myoblast
Figure 2. Schematic illustration of crosstalk between the oste- fusion to form muscle fibres and trophoblasts to build the
ogenesis and osteoclastogenesis – bone remodelling – process outer layer of a blastocyst (Anderson, 2000). The osteoclas-
Reprinted with permission from (Bassett et al., 2003). tic development process involves attachment to the bone
Copyright © 2003 Elsevier
surface, migration and subsequent degradation of bone
matrix components, whereas osteoclast cells interact with
vascular endothelial growth factor (VEGF), expressed by typical extracellular protiens, e.g. fibronectin, collagen,
downstream hypoxia-inducible transcription (HIF)-1α in bone sialoprotein and osteopontin (Faccio et al., 1998;
osteoblasts to enhance vascularization, seems to be also Grano et al., 1994). Figure 3 illustrates the different steps
relevant for osteoclast behaviour (Deckers et al., 2000). of osteoclastogenesis.
Results have shown that VEGF influences osteoclasts and An individual cell can have different membrane compo-
plays an important role in bone development, angiogene- nents and cytoskeletal structures in different parts of the
sis and tumour metastasis (Yang et al., 2008). Beside the cell. This is called ’cell polarization’ and four major mem-
role of VEGF type-A in osteoclasts, other studies have brane domains occur in this active stage, which can be
shown that VEGF type-C increases the resorption activity characterized by laser scanning confocal microscopy and
of osteoclasts (Zhang et al., 2008; Trebec-Reynolds et al., scanning electron microscopy (SEM) (Salo et al., 1996).
2010). Furthermore, it seems that VEGF-C is a kind of The ruffled border membrane is next to the substrate
’new RANKL target gene’ in osteoclasts. Indeed, VEGF which is being resorbed; the sealing zone area is
can be seen as an autocrine factor regulating osteoclast connected by the cell attachment to the substrate; and
activity. As described in this paragraph, therefore, the reg- the periphery and central domain define the basal surface
ulation of bone remodelling during development, fracture (Lakkakorpi and Vaananen, 1991; Salo et al., 1996).
repair and pathological bone loss is affected by several Typical examples for cell–cell contact (A), filopodia (B)
signals (M-CSF, RANKL and VEGF). and different structures of the cell membrane (B–D) are
shown in Figure 4.
The resorption of bone tissue is a cascade of processes
during which an osteoclast migrates to the resorption site,
3. Description of the development of attaches to the bone surface, dissolves both inorganic and
osteoclasts in general organic phases of the bone matrix, detaches and moves to
another resorption place or undergoes apoptosis
When discussing osteoclastic activity, usually osteoporo- (Holliday et al., 1997; Roodman, 1996; Blair, 1998). The
sis, bone infection and osteolysis are considered (Kong movement of osteoclasts includes changes in cytoskeleton,
and Penninger, 2000). Moreover, osteoclasts can also polarity and function. The determination of the actual
phagocytose particles of a wide range of sizes, e.g. state of activation can be observed by morphological and
Figure 3. Illustration of differentiation steps of monocytes (here colony-forming unit–macrophage, CFU-M) into osteoclast cells.
Reprinted with permission from Takahashi et al. (1999). Copyright © 1999 Elsevier
DOI: 10.1002/term
Figure 4. SEM-images of different cell membrane compartments of stimulated RAW 264.7 cells. (A) Cell fusion; arrow shows plasma
membrane fusion by actin bridges. (B) Cell attachment; arrows indicate the flat edge with filopodia. (C) Cell membrane with highly
structured basal membrane around the cell body. (D) Cell polarization on top of the cell with thin and flat shapes. Reprinted with
permission from Detsch et al. (2009). Copyright © 2009 Mensch und Buch Verlag, Berlin
cytoskeleton changes (Lakkakorpi et al., 1989). The inter- an acidic pH value of 4.0–4.5 is essential for solubilization
action between osteoclast cells and the bone matrix is me- of the hydroxyapatite mineral (Väänänen et al., 2000;
diated by dot-like podosomes, which are focal adhesions Gregory et al., 1974). The basic cellular mechanism respon-
containing at least F-actin, talin and vinculin (Lakkakorpi sible for this acidification is the active release of protons
and Vaananen, 1991). Mature osteoclast cells engage in into the extracellular space. These protons dissolve hy-
resorption via peripheral attachment to the matrix by droxyapatite crystals and both cations and anions (Ca2+
the α5β3 integrin, which creates a microcompartment or PO4 3- ions) are removed. For the extracellular acidifica-
between the ruffled basal border of the cell and the bone tion, carbonic anhydrase type II (CA II), a family of cyto-
surface. When the cell starts to polarize for resorption, solic enzymes, is needed (Lehenkari et al., 1998). CA II
these dots are grouped to form a disk-like pattern, which catalyses the conversion of carbon dioxide and water to
then becomes encircled by a single actin ring and a H2CO3. In this context, a direct modulator of osteoclastic
double vinculin ring inside the cell. Interestingly, the bone resorption is the extracellular Ca2+ ion level (Seuwen
sealing zone is a highly dynamic structure which needs et al., 1999). Ca2+ ion concentrations in the resorbed area
adhesion-mediated formation (Anderegg et al., 2011). rise from 8 to 40 mM. These levels are indeed ’physiologi-
These rings are components of the sealing zone and their cal’ for active osteoclasts, which seem suitable for handling
appearance can be used as a marker for attached osteo- such high Ca2+ ion loads (Zaidi et al., 1999). The ruffled
clasts (Väänänen, 2005). The sealing zone area is tightly border membrane has been shown to be enriched in vacu-
attached to the bone surface, separating the ruffled border olar-type proton ATPases, which are structurally related to
area from the rest of the cell membrane. Then, resorption kidney H+-ATPase, but it differs from mitochondrial and
of hydroxyapatite from bone is caused by the ability of os- gastric proton pumps. Figure 5 illustrates an active osteo-
teoclasts to generate an acidic extracellular compartment clast in cross-section (Stenbeck, 2002). During the last
between the cell and the bone surface. Here, pH values steps of resorption, lacuna formation and actin and
of up to 3 or less occur under the ruffled border (Baron, vinculin rings disappear and the cell is ready to move and
et al., 1985; Blair et al., 1989), which has been measured start a new resorption cycle. The organic and inorganic
by microelectrode technique (Silver et al., 1988). Indeed, degradation products are endocytosed from the ruffled
DOI: 10.1002/term
progenitor cell line (Miyamoto et al., 1998). By stimula-

tion with macrophage colony-stimulating factor (M-CSF)
and the receptor activator of the NF-κB ligand (RANKL),
these cells are utilized for in vitro osteoclastogenesis and
remodelling studies (Kanatani et al., 2003). RAW 264.7
cells (a mouse leukaemic monocyte–macrophage cell line,
established from the ascites of a tumour induced in a male
mouse by intraperitoneal injection of Abselon leukaemia
virus) could also be stimulated with M-CSF and RANKL.
The RAW 264.7 murine cell line has proved to be an im-
portant tool for in vitro studies of osteoclast formation
and activation (Wei et al., 2001). When RAW 264.7 mono-
cytes were stimulated with RANKL and M-CSF, they
formed multinucleated giant cells after 14 days of cultiva-
tion. Typical cell morphology changes from the attach-
ment of a monocyte to the formation of a giant cell are
Figure 5. Cross-section of a bone resorbing osteoclast with an shown in Figure 6 (Detsch, 2009). The morphology of
overview of the major pathways 1–9: moving of vesicles, acidifi- osteoclast-like cells attached to HA was flat and round
cation of extracellular space, carbonic anhydrase generates pro-
and exhibited many pseudopodia and filopodia. At the
tons, chloride–bicarbonate exchange, endocytosis of organic
matrix, transcytosis of endocytosed matrix proteins, receptor- beginning of the cell–material interaction, small round
mediated endocytosis from basolateral plasma membrane and monocytes adhered to the surface, then these cells devel-
recycling vesicle from the exit site. Reprinted with permission oped an edge, fused together and exhibited osteoclast-like
from Stenbeck (2002). Copyright © 2002 Elsevier cell morphology.
It can roughly be estimated from in vitro resorption
border membrane. Afterwards, they are transported in assays with osteoclasts isolated from 2 day-old rats that
vesicle-like structures through the cell to the plasma mem- the formation of a resorption lacuna takes 4–48 h (Detsch
brane domain and released from there into the extracellu- et al., 2005). However, it must be noticed that these
lar space (Salo et al., 1996, 1997). values can vary considerably, depending on the resorption
activity of the cells as well as on the area and depth of the
resorption lacuna. Currently, to the authors’ knowledge,
osteoclasts cannot be synchronized in in vitro cultures
4. In vitro osteoclastogenesis and, as there is no established system or ISO norm to an-
alyse osteoclast behaviour on biomaterials, it is difficult,
4.1. Cells and in most cases impossible, to quantitatively compare
the different osteoclastic resorption models investigated.
Of particular relevance for bone tissue engineering, active While cell lines are a useful tool for examining resorption
osteoclast cells start the remodelling process by generat- behaviour, it should also be mentioned that with primary
ing space for osteoblasts and endothelial cells, which are cells, especially primary monocytes, it is possible to study
necessary for the formation of new autologous bone the whole in vitro osteoclastogenesis process. Neverthe-
tissue. To study the role of osteoclast cells in bone tissue less, the RAW 264.7 cell line is widely used for model
engineering, the source of cells has first to be defined. osteoclastogenesis studies in vitro. Dentine and cortical
There are several biological methods to generate osteo- bone slices have been reported to be substrates for observ-
clast cells for in vitro studies. Sources for osteoclastic pre- ing the general development of osteoclastogenesis in vitro
cursor cells are bone marrow and peripheral blood (Cody et al., 2011; Boyde et al., 1984; Kakudo et al., 1996).
mononuclear cells (PBMCs), which can be stimulated to Moreover, these surfaces, as natural surface models, are
differentiate into resorbing cells (Severson, 1983). Even also appropriate assays for studying the effects and thera-
human mononuclear leukocyte suspensions isolated from peutic profiles of drugs such as bisphosphonates on osteo-
umbilical cord blood can also be differentiated to osteoclasts (Zhang et al., 2012; Susa et al., 2004).
clast-like cells (Fong et al., 2013). Furthermore, osteoclast
cells can be isolated from native bone tissue, e.g. new-
born rats (Detsch et al., 2005; Monchau et al., 2002). Cell 4.2. Parameters to identify osteoclasts and their
lines such as human U-937 (leukoma monocytes) can also activity on biomaterials
be stimulated to become active resorbing cells (Blottière
et al., 1995). These cells, cultured in a standard culture Due to the fact that there is no specific single osteoclast
medium, can grow in suspension and display spontaneous cell marker, different parameters should be analysed to-
adhesion. With the addition of vitamin D, 1α,25(OH)2D3 gether. Enzymes such as acid phosphatase (AP) are local-
and phorbol-12,13-dibutyrate to the culture medium, cell ized in nearly all cell types. They are stored in lysosomes
differentiation of U-937 cells to giant cells occurs. C7 is and function when these fuse with endosomes. However,
the name of another mouse macrophage-like osteoclast AP is a typical (non-specific) marker of monocytes and
DOI: 10.1002/term
Figure 6. Development of osteoclast-like cells on a hydroxyapatite surface (SEM images of stimulated RAW 264.7 cells after different
incubation times). The morphology changes from monocytes to large osteoclast-like cell forms. Reprinted with permission from
Detsch et al. (2009). Copyright © 2009 Mensch und Buch Verlag, Berlin
macrophages, while tartrate-resistant acid phosphatase

(TRAP) is highly expressed by osteoclasts, activated mac-
rophages and neurons. Especially, the isoform TRAP 5b,
which can be detected in serum, seems to be a specific
marker for osteoclast activity (Halleen et al., 2000;
Habermann et al., 2007). In osteoclasts, TRAP is localized
within the ruffled border area, in lysosomes, Golgi cister-
nae and vesicles. Therefore, TRAP staining is a commonly
used method to differentiate between TRAP-positive
osteoclast-like cells and undifferentiated monocytes (Faust
et al., 1999; Connor et al., 1995). To illustrate TRAP-positive
cells, TRAP-stained cells, originated from PBMCs after 28
days of cultivation are shown in Figure 7. Additionally,
vitronectin receptor (VNR), RANK receptor (which binds
RANKL) and calcitonin receptor (CTR) are further markers
that have been used to describe osteoclastic cell behaviour
Figure 7. LM-image of TRAP-stained osteoclasts originated from
(Väänänen et al., 2000; Roodman, 1996; Blair, 1998).
PBMCss after 28 days of stimulation with M-CSF and RANKL.1
The morphological change of monocytes into osteoclast-
like cells documents in vitro osteoclastogenesis. It is thus the culture medium and especially in an undefined
relevant to discuss how the resorption activity can be char- content in fetal calf serum (FCS). The functional activity
acterized. One possible method is to determine the resorp- of osteoclast-like cells can also be determined from
tion products in the supernatants of the cultures. However, topographical changes to the test surfaces. To examine
in the case of calcium and phosphate ions, as main resorp- the role of biomaterial surface properties in resorption
tion products of bone apatites or calcium phosphate lacuna formation, different methods can be applied for
ceramics, measurement by inductively coupled plasma
mass spectrometry, for example, can lead to errors, consid- 1
PBMCss were provided by Professor E. Strasser, Department of Trans-
ering that calcium and phosphate ions are also present in fusion Medicine and Haemostasiology, University Hospital Erlangen.
DOI: 10.1002/term
3D volume characterization of lacunae (Goldberg et al., monocytes can be stimulated by M-CSF and RANKL to
2012; Pascaretti-Grizon et al., 2011). Methods to deter- differentiate into multinucleated cells, and HA can be
mine the resorption activity are related to the survey of resorbed by osteoclast-like cells.
the formed lacunae by different microscopic techniques
(LM, RAMAN, CSLM, and SEM). Additionally, μ-RAMAN-
microscopy with confocal optics, autofocus systems and data
analysis software can be employed to generate 3D images.
5. Different material parameters
The resorption activity of osteoclasts can be quantified by influencing osteoclastogenesis
measuring number, diameter distribution, depth and volume
of the lacunae. In addition to material Detsch et al. (2011) To optimize the activity of osteoclasts for bone tissue engi-
were able to calculate the resorption activity on BCP surfaces neering, it is essential to know how different material
by characterizing the dimensions of lacunae: depths of parameters affect the response of osteoclasts. Numerous
10–12 μm and diameters of 30–40 μm were measured material properties and characteristics have been investi-
(Detsch et al., 2011). The resorbed volume amounted from gated in this area (Shimizu et al., 1989; Doi et al., 1999;
ca. 170 μm3 for small acidifications to ca. 3000 μm3 for large Monchau et al., 2002; Duplat et al., 2007; Perrotti et al.,
lacunae. As an example, Figure 8 shows a SEM image 2009). In the following paragraph, the influence of differ-
exhibiting a lacuna on β-TCP ceramic. The resorbed area is ent materials and material modifications on the differenti-
characterized by an ‘intercrystalline structure’, which seems ation and activation of osteoclasts is discussed. As
to have proceeded along the grain boundaries, and the sur- described, bone cells are involved in bone healing follow-
face of the TCP grains was partly resorbed, remaining in the ing bone trauma. Therefore, bone replacement materials
form of small spikes (Figure 8, right) (Detsch et al., 2008b). must be able to provide support for both osteoclasts and
In comparison, resorption studies on dentine slices, osteoblast functions in order to ensure the dynamics of
where the depth and volume of resorption per lacuna bone remodelling. There is a clear relationship between
were measured, have shown the depth to be in the range the chemical composition of the sample surface and the
9–30 μm and the volume of resorption/lacuna to be cell morphology of the resorbing cells (Pierce et al.,
3000–60 000 μm3 (Mabilleau et al., 2012). Compared to 1991). Osteoclastogenesis is influenced, amongst other
the results achieved on HA, lacunae on dentine were parameters, by the percentage of mineralization of the tis-
much deeper and more volume was resorbed. Osteoclast sue. For example, the resorption of enamel (95%) was
acid secretion into the sealed extracellular substratum shown to be lower than that of dentine (70%) and cemen-
causes an ion release from the calcium phosphate surface. tum (65%), but the dentine and cementum lacunae were
Osteoclasts are multinucleated cells and the major effect not significantly different from each other (Jones et al.,
of cell fusion of monocytes is obviously to increase the cell 1995). The sizes of the formed lacunae correlated with
size of osteoclasts, which enables them to resorb larger the relative mineral densities of the three tissues, showing
areas of bone tissue. The single activity of an osteoclast that the rate of osteoclastic activity is inversely propor-
cell can be determined by quantification of the formed tional to mineral density. Taylor et al. (2002) analysed
lacunae. In this context, the diameter, depth and volume the cell behaviour of neonatal rabbit osteoclasts on 11 dif-
of the lacunae depend on the amount of nuclei in the ferent bone substitute materials. The materials used were
osteoclast. Ishii and Saeki (2008) assumed that the cell bone-derived, including freeze-dried human rib block, hu-
(both osteoclasts and their precursor mononuclear cells) man demineralized freeze-dried bone and deproteinated
is round and can resorb a hemisphere whose cross-section bovine bone, in addition to synthetic HA, coated methac-
is identical to the cell size. These results indicate that rylates and coated silica glass. While most of these
Figure 8. SEM images of β-TCP (cleaned surfaces) after 21 days of osteoclast-like cell cultivation: lacuna (left) and resorbed TCP
grains at higher magnification (right). Reprinted with permission from (Detsch et al., 2008b). Copyright © 2008 Elsevier
DOI: 10.1002/term
materials supported the adhesion of osteoclasts, only the formation. However, the optimal degradation rate and
bone-derived HA materials displayed resorption lacunae the mechanisms of degradation are controversially
and the non-HA materials showed no pit formation. discussed in the literature for different materials and
Beside the chemical composition of the sample, surface systems. Regarding this application, the chemical disso-
energy (wettability) also plays an essential role in osteo- lution of some biomaterials, e.g. TCP, is usually faster
clast adhesion. Cells are usually seen to adhere signifi- than osteoclastic resorption. On the other hand, most
cantly less well to low-energy surfaces in comparison to natural bone graft substitutes and HA ceramics undergo
HA surfaces with much higher surface energy values very little chemical dissolution and their resorption is
(Redey et al., 1999). primarily by cellular mechanisms (Taylor et al., 2002;
Beside material composition, also roughness, crystallin- Detsch et al., 2008b).
ity grain size and other specific material properties, e.g. Nanocrystalline calcium phosphates containing carbon-
the dissolution rate of a material, can inhibit or enhance ate have a high similarity to bone mineral. The reactions
bone resorption. Typical calcium phosphate ceramic mate- of bone cells (primary osteoblasts and osteoclast-like
rials used in bone tissue engineering are β-tricalcium cells) on these materials in comparison to sintered β-TCP
phosphate (TCP) and hydroxyapatite (HA). Both ceramics and HA have confirmed the biocompatibility of the nano-
are highly biocompatible and osteoconductive (Durrani crystalline samples (Detsch et al., 2010). However, osteo-
et al., 2010). In contrast to HA, TCP ceramics have a higher clastic differentiation was constrained on carbonate-rich
degradation rate (chemical dissolution). The increasing samples, leading to a small number of osteoclast-like cells
Ca2+ ion concentration in culture medium, caused by the on the materials, and only few resorption pits were
higher dissolution of TCP compared to that of HA, inhibit formed. The grain size of the calcium phosphate ceramics
osteoclast develompment (Detsch et al., 2008b). HA is (nano- vs micro-) was less important than expected from
more stable and undergoes only a slow resorption (Roldán physicochemical considerations. When comparing the
et al., 2010). It is interesting to note that the impact of different nanocrystalline samples, the highest resorption
bone morphogenic protein (BMP), a growth factor to in- rate was found for nano-HA with low carbonate content,
duce bone formation, on the resorption and degradation which strongly stimulated the differentiation of osteo-
of a calcium phosphate ceramic seems to be quite high. clast-like cells on its surface. The influence of macroscopic
In an implantation study with BMP-loaded calcium phos- and nanocrystalline calcium phosphate ceramics has been
phate ceramics in an ectopic minipig model, BMP-7 was studied recently with human monocytes after 23 days of
found to support bone formation and additionally to affect osteoclastic stimulation (Despang et al., 2013). The
ceramic degradation (Roldán et al., 2011). In another results revealed an increased expression of osteoclast-
study (Yamada et al., 1997), the influence of the phase relevant genes, such as TRAP, vitronectin receptor and
composition on physicochemical dissolution and osteo- cathepsin K, which was at the same level for all mate-
clastic resorption of biphasic calcium phosphate ceramics rials examined. Another parameter suggested to tailor
was demonstrated. With a higher β-TCP fraction the disso- osteoclastic activity is the grain size (GS) of the
lution rate of the ceramics increased due to the higher bioceramic. To investigate the effect of GS, almost dense
solubility of the β-TCP phase. The most pronounced devel- HA–ceramics with three different grain sizes (2, 7 and 14
opment of osteoclast-like cells was found on ceramics with μm, labelled GS2, GS7 and GS 14, respectively) were
HA:β-TCP ratios of 100:0 and 80:20 (wt%). The cells on prepared and analysed using stimulated RAW 264.7 cells
these ceramics were multinucleated and had a larger cell (Detsch, 2009). The results showed that the grain size of
size than on the other bioceramics investigated. On all HA–ceramics had a decisive influence on their degrada-
ceramics except pure β-TCP, lacuna formation could be tion behaviour (chemical dissolution and resorption).
detected as a result of resorption. It was claimed that for Differentiation analysis by RT–PCR showed the expres-
integration in the physiological bone remodelling sion of osteoclast cell markers, such as TRAP 5 and
process, biphasic calcium phosphate ceramics with a carbonic anhydrase type II, on all HA–ceramics. How-
HA:β-TCP ratio of 80:20 and 60:40 are the most appropri- ever, cathepsin K was not found in cells cultured on
ate, because of their adequate chemical dissolution and HA–ceramics with 14 μm grain size, which showed that
positive influence on the development of osteoclast-like precursor osteoclasts could also resorb inorganic
cells (Mayr et al., 2008). With respect to both degradation surfaces without expression of an essential osteoclast
mechanisms, HA and BCP seem to be ’better’ for bone marker such as cathepsin K. As a consequence, the per-
remodelling than TCP, which, in fact, showed a higher formance of calcium phosphate-based materials in bone
dissolution than HA and a comparable development of tissue-engineering approaches can be optimized by
osteoclast-like cells, but no resorption. Within the variation of the grain size (Detsch, 2009). Another
efforts of bone-regeneration strategies towards restitutio important parameter to be considered is the surface
ad integrum, bone-filling materials or scaffolds that are roughness and its effect in stimulating osteoclast-like
implanted for the substitution of large bone defects cells. For osteoblasts it is well known that the biomate-
should be completely degraded within an adequate rial surface roughness has a strong influence on initial
period of time. A too fast a degradation rate will not cell behaviour (Anselme et al., 2000; Lincks et al.,
allow sufficient time in which bone can grow, whereas 1998). The initial cell reaction is important for bone
a too fast degradation rate can inhibit new bone ingrowth into implanted materials or scaffolds, and
DOI: 10.1002/term
therefore the surface roughness effect is relevant for tissue on the sol–gel-derived bioactive glass AP40 (Cacciotti
engineering. In contrast to the well-investigated reaction et al., 2013). Osteoclast behaviour was also analysed on
of osteoblasts to different surface structures, the influ- sol–gel-derived bioactive glasses (SG600–SG1100) with
ence of surface roughness on osteoclast activity has different crystallinity (Zhao et al., 2005). Interestingly,
rarely been researched. In the previously mentioned resorption lacunae were found on all surfaces. Accordingly,
study (Detsch, 2009), the roughness (Ra)of HA surfaces Karpov et al. (2008) also detected osteoclastic activity on
ranged from Ra = 0.02 [polished (POL)] and Ra = 0.18 sol–gel-derived bioactive glasses. Other in vitro studies
[as-fired (AF)] to Ra = 1.45 μm [sandblasted (SB)], have shown that bioactive glasses and apatite- and wollas-
whereas the grain size was 3 μm after sintering for all tonite-containing glass–ceramics (A-W GCs) can bind to
samples. Cell biological analyses showed, above all, that bone chemically through a bone-like apatite layer formed
the differentiation of monocytes into osteoclast-like cells on their surfaces after implantation into a bone defect.
was inhibited by the rough SB surface, although forma- This resulting surface layer is composed of carbonate
tion of small lacunae was detectable on these surfaces. containing hydroxyapatite similar to bone mineral
It was shown that a smoother surface enhanced osteo- (Hench, 1998). Carbonated hydroxyapatite, in an amor-
clast formation to a higher extent than a rougher one phous or nanocrystalline state, can be resorbed by osteo-
(Detsch, 2009). This result can be explained by the opti- clasts and can be integrated in the bone-remodelling
mal possibility for cell spreading caused by the low process (Detsch et al., 2010; Yamada et al., 1994;
roughness. Furthermore, strongest osteoclastic differen- Leeuwenburgh et al., 2001). Figure 9 illustrates the
tiation was found on the AF topography, which indicates hypothesis of osteoclast interaction with bioactive glass
the positive influence of small structures. Similar results surfaces. Vaahtio et al. (2006) investigated different
were shown using HA samples with Ra of 0.04–0.58 μm. silica- and carbonate-containing calcium phosphate
Other authors have shown that the activity of human os- layers on bioactive glasses. Active osteoclasts were found
teoclast precursor cells increased with the surface rough- on the calcium phosphate layer instead of on plain
ness (Costa-Rodrigues et al., 2012). Limited related surfaces (Vaahtio et al., 2006). Silicate bioactive
studies have also demonstrated an enhanced osteoclast- glasses, e.g. 45S5 Bioglass®, also exhibit favourable
like cell function on ceramic surfaces with nanometer- characteristics for bone tissue-engineering applications
size surface topography (Webster et al., 2001). Gross (Chen et al., 2006; Fu et al., 2011). On this material
et al. (2012) found that the resorption of human-derived the velocity and level of apatite formation seems to
osteoclasts revealed a 10-fold greater number of resorp- influence osteoclastic differentiation and activation
tion pits on dentine and on thermally sprayed coatings (Bosetti and Cannas, 2005).There is, however, ex-
compared to polished coatings. These authors hypothe- tremely limited work published on the interaction of
sized that HA coatings with rougher topography un- osteoclast cells and bioactive glass and glass–ceramic
dergo active remodelling, like dentine. 3D scaffolds, this being identified as an area requiring
Beside CaP based ceramics, materials based on silicate further research effort.
glasses and glass–ceramics are also being extensively in- Apart from basic CaP or silicate material composition, me-
vestigated as bone substitute materials and bone scaffolds tallic ions, e.g. strontium (Sr), zinc (Zn), boron (B), copper
(Chen et al., 2006; Juhasz and Best, 2012; Fu et al., 2011; (Cu), cobalt (Co), vanadium (V), lithium (Li) and magne-
Bielby et al., 2004). Bioactive glass, e.g. 45S5 Bioglass®, is sium (Mg), are becoming increasingly interesting in bone tis-
an amorphous inorganic material containing calcium, sue engineering approaches because they are known to be
sodium, phosphor, silicon and oxygen, which in bulk form involved in bone remodelling, angiogenesis and mineraliza-
exhibits a slow chemical dissolution rate. In vivo studies tion of bone tissue (Hoppe et al., 2011; Mouriño and
have shown that bioactive glasses are degradable by a com- Boccaccini, 2010). For example, Cu, Zn, Sr and, further-
bination of cellular mechanisms and chemical dissolution more, fluoride and carbonate, have been shown to lead to
(Oonishi et al., 1997; MacNeill et al., 1999). Another decreased osteoclastic activity on rabbit osteoclasts in a
in vivo study evaluated the resorption of three different dose-dependent manner (Yang et al., 2010). In the scope
types of bioactive glass (Vogel et al., 2001). The authors of biomaterials development for tissue engineering, bioac-
concluded that 45S5 bioactive glass displayed the highest tive glasses and calcium phosphate ceramics can be doped
bone-bonding kinetics as well as degradation rates. In with metallic ions (Sr is widely used) to influence osteo-
terms of osteoclast action on bioactive glasses in vitro, clast cell behaviour (Roy and Bose, 2012; Gentleman
results are rare. One investigation was carried out on et al., 2010). Mostly these approaches are aimed at
S53P4 bioactive glass (Wilson and Trumpp, 2006). On inhibiting resorption activity of osteoclasts and supporting
this material, cultured osteoclasts could not form any re- osteogenesis, e.g. the addition of strontium in HA thin films
sorption lacunae. In this context, it seems that released obtained by pulsed-laser deposition enhanced the osteo-
Si inhibits the development of RAW 264.7 cells directly genesis of MG-63 cells and inhibited the activation of
(Mladenović et al., 2014), e.g. large amounts (> 25 μg/ human osteoclasts (Capuccini et al., 2008).
ml) of added Si to the cell culture medium were found The interaction of osteoclast cells with other biomate-
to induce a reduction of RANK expression and therefore rials, including metals and biopolymers, has been studied
a decrease of in vitro osteoclastogenesis. In addition to a limited extent. Although metals such as titanium and
PBMCs have been shown to differentiate into osteoclasts its alloys are non-degradable, osteoclast behaviour has
DOI: 10.1002/term
Figure 9. Schematic progress of osteoclast cell activation and lacuna formation on bioactive glass surface: bone-like layer formation
on the surface, cell attachment of precursor cells and osteoclastogenesis (cell fusion and differentiation), formation of ruffled border
and resorption of the bone-like layer, lacuna formation and, finally, new apatite formation with the same thickness
been investigated by cultivation of RAW 264.7 cells, show- inorganic materials (Lees et al., 2001). One example in
ing osteoclastic differentiation (for a recent study, see the field of hydrogels is the incorporation of cathepsin K-
Brinkmann et al., 2012). In the field of biodegradable sensitive peptides into the polyethylene glycol (PEG) bio-
metal implants, magnesium alloys are in the focus of polymer backbone to activate hydrogel resorption by osteo-
research for possible bone substitution applications (Song, clasts (Hsu et al., 2011). In this study the authors reported
2007; Witte et al., 2008). In a relevant in vivo study it was that RAW 264.7 cells gradually degraded the material with
shown that the corrosion products of magnesium alloys cathepsin K secretion. RAW 264.7 cells were also used to an-
decreased osteoclastic activity, although bone formation alyse the influence of polymeric phosphate/polyphosphate
was enhanced (Janning et al., 2010). It should be also (polyP) at different concentrations (Wang et al., 2012).
pointed out that although biopolymers are a promising The highest inhibition of TRAP-positive cells was deter-
class of materials potentially useful in bone regeneration, mined at 1.0 μM polyP compared to the reference with no
there are only a few studies dealing specifically with the supplements. In a further study (Jones et al., 2009), mono-
interaction of osteoclasts with polymers used in bone tissue cytes were isolated freshly from 8 week-old CD1 mice and
engineering. In a relevant recent study, unmodified and cultivated with M-CSF and RANKL on fibroin, poly(L-lactic
fibrinogen-modified chitosan substrates were used to ana- acid) (PLLA) and chitosan for 10 days. The results showed
lyse the formation of multinucleated osteoclasts (Torres that TRAP-positive cells were placed on fibroin and chitosan
et al., 2013). The results showed a significantly higher num- and smaller numbers of TRAP-positive cells were found on
ber of highly multinucleated osteoclasts (> 10 nuclei/cell) PLLA. In another study, polycaprolactone (PCL), another
on modified chitosan compared to chitosan alone (Torres synthetic biopolymer widely considered for bone tissue scaf-
et al., 2013). Collagen, another natural polymer used in folds (Hutmacher et al., 2001), was coated with a biomi-
bone-regeneration applications, has also been considered metic HA layer by immersion in simulated body fluid and
in relation to osteoclast activity; it is well known that afterwards osteoclasts, isolated from the long bones of neo-
osteoclasts can degrade collagen by cathepsin K and natal New Zealand white rabbits, developed their typical
metalloproteinase (Väänänen and Laitala-Leinonen, 2008; phenotype (Costa et al., 2013). In contrast to work investi-
Hou et al., 2001; Delaissé et al., 2003). Shafieyan et al. gating HA-coating of PCL, Kadow-Romacker et al. (2013)
(2012) showed osteoclastic activity on collagen-coated recently coated β-TCP with poly(D,L-lactide) (PDLLA) and
polydimethylsiloxane. Relevant in vitro studies have also loaded the polymer with different concentrations of
been performed on composites combining collagen and zoledronic acid. Afterwards, the behaviour of osteoblast
DOI: 10.1002/term
and osteoclast cells on several material combinations was number and differentiation potential with increasing age
studied. Interestingly, osteoclast cell viability and TRAP ex- (Stolzing et al., 2008). More recently, umbilical cord
pression were significantly decreased after cultivation on blood (UCB), attainable by a non-invasive method, was
PDLLA-coated β-TCP, with or without zolendric acid, for introduced as an alternative source for MSCs (Majore
12 days (Kadow-Romacker et al., 2013). Investigations with et al., 2011). Another promising source of MSCs is adipose
osteoclasts (stimulated progenitor cells from bone marrow) tissue (AT) (Chamberlain et al., 2007, Goodwin et al.,
on poly(3-hydroxybutyrate-co-3-valerate) (PHBV) and on 2001). The kind of cells used and the reaction of the cells
PHBV with HA particles showed no actin ring formation in with the scaffold material are also influenced by the deg-
the cells and therefore no resorption on these surfaces radation behaviour of the scaffold. This is because bone-
(Cool et al., 2007). The results discussed in this section substitute materials that are implanted for the replace-
thus show that osteoclasts, beyond resorbing inorganic ment of larger bone defects should be completely
biomaterials, can also interact with biopolymers. Indeed, degraded within an adequate period of time. The degra-
in bone tissue-regeneration strategies using organic dation process of scaffolds is regulated by two mecha-
substrates, resorption seems to depend not only on macro- nisms: (a) dissolution due to the intrinsic solubility of
phages but also on osteoclasts (Torres et al., 2013). the biomaterial phase, including e.g. calcium phosphates,
Clearly, much more dedicated research effort is needed different types of biodegradable polymers or bioactive
to further fully understand the interactions between oste- glasses and even biodegradable Mg alloys; and (b) cellu-
oclasts and biodegradable polymers, and to elucidate the lar degradation by osteoclasts, also called resorption.
effect of cellular activity on polymer degradation kinetics Besides patient-related factors (defect localization, defect
by hydrolysis or enzymatic mechanisms. size, age, gender and health situation), it is recognized
that degradation of scaffolds is mainly influenced by the
composition of the material and the geometry of the scaf-
fold, meaning not only the overall scaffold size but also its
6. Bone tissue engineering (BTE) micro- and macroporosity. As it is well known, calcium
phosphate ceramics such as hydroxyapatite and tricalcium
6.1. General aspects of bone tissue engineering phosphate are among the most-implanted biomaterials in
this field. These CaP materials have excellent biocompati-
Over the past few years BTE has undergone considerable bility because they are very similar to the inorganic phase
progress, demonstrating great potential for the recon- of human bone (Olsen et al., 2000, Deisinger et al., 2008).
struction of damaged bone in comparison with conven- One general characteristic of bone substitute materials for
tional therapies (Panetta et al., 2010). For large bone bone-regeneration applications is that they must be
defects, conventional autologous bone graft is currently resorbed by osteoclast cells and replaced by new bone. In-
the most-applied therapy. Challenges in the fabrication deed, it is essential that all bone-substitute materials are
of synthetic bone scaffolds include achieving suitable me- included in anabolism or tissue formation and in catabo-
chanical stability, realisation of a high, 3D interconnected lism or remodelling (Detsch et al., 2008a, 2011). In addi-
porosity and optimization of pore geometry for bone tion to the experimental results of remodelling processes
ingrowth and vascularization. Currently, commercially discussed above, the dynamic behaviour between osteo-
available synthetic bone substitute materials are offered clasts, osteoblasts and the biomaterial is also proposed
in distinct geometries and sizes (blocks, cylinders, to be further studied using bioinformatics. Currently,
wedges) with a certain porosity. The external geometry there are different mathematical models available to de-
can only be changed by machining the block in the operat- scribe bone remodelling (Pivonka and Komarova, 2010).
ing theatre, which is inconvenient, increasing the costs Moreover, mathematical models can also be useful to
and possibly resulting in insufficient fitting accuracy. Fur- examine in detail the interactive roles of osteoblasts and
thermore, it is usually not possible to readily adjust pore osteoclasts, namely autocrine and paracrine regulation
size and geometry or total porosity to the requirements (Komarova et al., 2003). Modelling of the interactions of
of the defect. In the context of BTE, besides combinations osteoblasts and osteoclasts, even in a simple form, requires
of bioactive scaffolds and several growth factors, living great computational effort, given that the communication
cells are investigated to optimally yield functional bone of these two cell types results in highly complex and non-
tissue for implantation purposes (Ringe et al., 2002). In linear behaviour. Furthermore, computational models indi-
an early publication, Bruder et al. (1994) state that cate that the intrinsic properties of the system can give rise
human mesenchymal stem cells (MSCs) can regenerate to the complex modes of bone remodelling observed in vivo.
bone in a clinically significant osseous defect and may In this context, materials such as calcium carbonate,
therefore provide an alternative to autogenous bone hydroxyapatite (HA), bioactive glasses (BG), tricalcium
grafts (Bruder et al., 1994). MSCs currently represent a phosphate (TCP), biphasic ceramics of HA and α- or β-
promising tool for clinical concepts in supporting regener- TCP, biopolymers and numerous biodegradable polymer–
ative therapy (Mauney et al., 2005). Bone marrow (BM) ceramic composites are of interest (Redey et al., 1999; Chen
was the first source reported to contain MSCs. However, et al., 2006). However, in clinical applications, it is at pres-
for clinical use BM may be detrimental, due to the highly ent impossible to choose the ’best’-suited material for the in-
invasive donation procedure and the decline in MSCs dividual patient based solely on information about the
DOI: 10.1002/term
materials’ degradability. Indeed, the degradation rates differentiation has been studied on 3D printed scaffold
(chemical dissolution) and resorption behaviour of these surfaces consisting of pure HA and β-TCP as well as on a
synthetic materials continue to be controversially discussed biphasic mixture of HA and TCP (60:40) to analyse
(Detsch et al., 2008c; Ghanaati et al., 2012). whether and how these 3D printed calcium phosphate
surfaces can be resorbed by osteoclast-like cells (Detsch
et al., 2011). In this regard, cell proliferation, differentia-
6.2. Bone tissue engineering with participation tion and activation were analysed using the monocytic
of osteoclasts cell line RAW 264.7. The results showed that osteoclast-
like cells were able to resorb calcium phosphate surfaces
Of relevance for bone tissue engineering is the use of 3D exhibiting grain sizes of 4.2 μm (HA), 4.6 μm (BCP) and
scaffolds instead of dense two-dimensional (2D) discs. 9.5 μm (β-TCP). Interestingly, differences in form and size
The comparison of 2D and 3D samples is useful to high- of the lacunae on hydroxyapatite and tricalcium
light the impact of the more complex, porous 3D samples phosphate were observed. Furthermore, it was shown
on the degradation mechanisms involving osteoclasts. that biphasic calcium phosphate ceramics, because of
Indeed, the effects of phase composition and additional their osteoclastic activation ability by expressing typical
material properties, especially micro- and macroporosity, markers such as integrin β3, integrin αv, CA isoform II,
on degradation behaviour are enhanced on a 3D scaffold, RANK, cathepsin K and also TRAP 5b, exhibit the most
as could be shown by studying the dissolution of different promising surface properties of the ceramics studied to
calcium phosphate compositions and the related resorp- serve as printed 3D bone-substitute scaffolds (Detsch
tion analysis (Schaefer et al., 2011). The attachment, et al., 2011). Moreover, BCP scaffolds (HA:βTCP 70:30
differentiation and resorption activity of osteoclast-like and 80% porosity) seeded with osteoclast progenitor cells
cells (C7 cell line macrophages from bone marrow) were in combination with human adipose-derived cells showed
recently investigated in 3D porous bioactive glass evidence of a 3D remodelling processes (Papadimitropoulos
scaffolds (Midha et al., 2013). The applied material was et al., 2011). The in vitro osteoconductive/osteoinductive
prepared by a sol–gel technique and was a 70S30C bioac- properties of several bone substitute materials have been
tive glass (composition, 70 mol% SiO2, 30 mol% CaO) extensively analysed, but the resorbability of these materials
with macropores of ca. 373 μm and interconnection diam- is often ignored. In a recent study, Lehmann et al. (2012)
eter of 172 μm. Further studies on the impact of different showed remodelling by the differentiation of osteoblast
pore sizes, scaffold porosities (and consequently different and osteoclast precursors on pure and Si-substituted HA
mechanical properties) in in vivo situations should be ceramics. In another study (Tortelli et al., 2009), a 3D
conducted in relation to osteoclast cell behaviour. To opti- bone model was developed by using scaffolds composed
mize their integration and functionality, scaffolds should of 67% Si-TCP and 33% HA/β-TCP and co-culturing mu-
be adapted to the individual and specific bone defect in rine primary osteoblasts with osteoclast cells. Interest-
terms of size and morphology. Rapid prototyping is a con- ingly, an increase of osteoblastic differentiation in the
venient manufacturing method to tailor materials to the 3D matrix supported earlier osteoclast differentiation.
3D geometry of the defect (Deisinger, 2010, Seitz et al., Establishing co-cultures presents some special chal-
2009, Hutmacher et al., 2001, Salgado et al., 2004). Espe- lenges in the context of cell numbers, the growth factors
cially, 3D printing allows the manufacture of implants and used and incubation time. Herein the mechanism of
scaffolds whose shape can be designed to fit the bone cell–cell contact and the crosstalk of osteoblasts and oste-
defect using anatomical information obtained from the oclasts seem to enhance biomaterial resorption activity
patient (Warnke et al., 2010). Recently, osteoclastic cell (Jimi et al., 1996). Figure 10 shows examples of different
Figure 10. LM image of TRAP-stained co-culture human PBMCss and SaOs-2 osteoblastic cells in a HA sample (left). SEM image of
ST-2 bone marrow cells and RAW 264.7 cells co-cultured in a porous BCP scaffold for 21 days (right)
DOI: 10.1002/term
co-cultures on dense HA discs and highly porous scaffolds. preoperatively from a blood sample together with mesen-
The light microscope image (Figure 10, left) presents one chymal stem cells (BMSCs) from bone marrow biopsies.
TRAP-positive stained osteoclast cell (stimulated PBMC) To our knowledge, this is a preliminary approach which
with surrounding osteoblasts after 21 days. In the SEM has not been investigated further. The question that conse-
image (Figure 10, right), osteoclast-like cells (stimulated quently arises is whether osteoclastic active areas can also
RAW 264.7 cells) and bone marrow stromal cells (ST-2 stimulate osteoblast-like cells and even endothelial cells.
cells) can be identified, beside the morphology, by the
structured cell membrane. As the osteoclastic cells possess
a highly structured membrane (see also SEM images in
Figures 4, 6), the cell membrane of the cultured bone 7. Conclusion and outlook
marrow cells is quite smooth. To establish a co-culture
for remodelling studies, different modifications were When considering cells for BTE, osteoblast-like cells and
tested; however, only with the growth factor supplements their precursors are normally the focus of interest. How-
M-CSF and RANKL were osteoclasts and osteoblasts ever, functional healthy bone needs osteoblastic and oste-
achieved together. Therefore, with this cell combination, oclastic cells and relies on their communication and
it seems that the osteoblastic cells do not have ’the concerted behaviour. It is recognized that the material
power’ to stimulate osteoclasts during 21 days (Detsch properties of a scaffold affect the osteoblastic response,
et al., 2008a). but the present review has revealed that little information
Besides ceramic scaffolds, composites such as is available regarding the effect of the mentioned proper-
biomimetically mineralized collagen I fibrils also promote ties on osteoclastic cell behaviour. In vitro studies with
the resorption and formation of new bone. It has been osteoclastic precursor cells on synthetic bone substitute
shown that active osteoclast-like cells can migrate materials have shown that specific parameters inhibit or
through scaffolds and resorb the material, whereas osteo- enhance resorption. In bioceramics, the phase and chem-
blasts proliferated, differentiated and synthesized new ical composition have significant influence on the men-
extracellular matrix, which was also mineralized tioned degradation. Crystallinity, grain size and density
(Domaschke et al., 2006). Furthermore, Nakagawa et al of the surface seem to have less significant effects on
(2004) were able to demonstrate that osteoblasts and osteoclastic activity than surface chemistry. In addition,
osteoclasts were successfully differentiated together from the topography of an implant or scaffold surface is defined
bone marrow on a mineralized polymer scaffold. As by the manufacturing process and can be tailored to influ-
mentioned above, Jones et al. (2009) showed osteoclastic ence the development and spreading of osteoclast cells.
development on chitosan and fibroin films. In this study Altogether, scaffolds for BTE should be optimized for oste-
they also cultivated MC3T3-E1 osteoblasts in a co-culture oblasts and osteoclasts, which are both relevant at the
for bone tissue-engineering studies. same interface of the biomaterial and the surrounding
Interestingly, Han and Zhang (2006) described that the tissue. In addition, limited studies based on co-cultures
absence of osteoclasts result in abnormalities of in vitro support the statement that osteoblasts and osteoclasts
bone formation. Therefore, they hypothesized that osteo- are both important in regenerative medicine approaches.
clast cell introduction will provide a solution to the critical It is recognized that osteoclasts control osteoblasts in dif-
problem in bone tissue engineering: ferent ways, depending on their stage of differentiation.
Besides the materials’ chemical compositions and their
The way of osteoclast introduction is the first challenge processing, which are important topics in scaffold devel-
in exploring its role in bone tissue engineering. In vivo opment for bone tissue engineering (Khademhosseini
studies have shown that maturation of preosteoclasts, et al., 2006), the enhancement of the biological perfor-
the cells just before fusion to form multinucleated mance of the tissue-engineered scaffold is also in the
cells, requires formation and accumulation of bone focus of extensive research effort (Pirraco et al., 2010).
matrix despite the independence of preosteoclast ap- For example, one approach to improve the angiogenesis
pearance and bone formation. Thus, the application of the constructs is to use co-cultures with osteoblasts
of preosteoclasts to biomineralized matrix for and endothelial cells (Unger et al., 2010). With sufficient
osteoclastogenesis is a physiologically relevant strat- vascularization, the ideal scaffold should provide initial
egy for osteoclast introduction. In conclusion, osteo- support for osteoprogenitor cells to deposit mineralized
clast introduction holds the promise of structural bone matrix; then it should be slowly resorbed at the
and functional improvement of tissue-engineered same time that newly formed bone grows. Co-cultures of
bone (Han and Zhang, 2006). osteoclasts and osteoblasts could be beneficial to the
microenvironment of a bone scaffold, given that osteo-
In order to achieve a guided, reliable formation of clasts are involved in functional bone regeneration, as
regenerated bone tissue, a concept of cell-based constructs mentioned above. Future research should address issues
with established bone remodelling pathways that control related to the effect of suitable osteoconductive and
tissue formation is necessary. The development of a kind osteoinductive materials, including CaP bioceramics as
of ’therapeutic osteoclastogenesis’ could be applied by pri- well as bioactive glasses, biodegradable polymers and
mary monocytes (PBMCs, precursor osteoclasts) harvested composites, on the behaviour of osteoclasts, focusing on
DOI: 10.1002/term
understanding the interactions between bone formation Acknowledgements

and bone resorption in contact with different biomaterials.
The calcium phosphate material synthesis and characterization
studies discussed in this review were performed by R. D. in
collaboration with Dr U. Deisinger, Dipl.-Ing. S. Schäfer and Dipl.
Conflict of interest Min. H. Mayr from the Friedrich-Baur Research Institute for
Biomaterials, directed by Professor G. Ziegler, University of
The authors have declared that there is no conflict of Bayreuth (Germany) and Dipl.-Ing. F. Uhl from BioCer
interest. Entwicklungs GmbH (Bayreuth).
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DOI: 10.1002/term

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JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE REVIEW

J Tissue Eng Regen Med 2015; 9: 1133–1149.

The role of osteoclasts in bone tissue engineering

Received 6 February 2013; Revised 18 September 2013; Accepted 20 October 2013

1. Introduction the replacement of diseased bone by tissue-engineered

Copyright © 2014 John Wiley & Sons, Ltd.

polymeric and metallic biomaterials, which have been

progenitor cell line (Miyamoto et al., 1998). By stimula-

macrophages, while tartrate-resistant acid phosphatase

understanding the interactions between bone formation Acknowledgements

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