Fisher John P. e.a. (ed.) Tissue Engineering
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9-8 Tissue Engineering
a consistent finding that the larger the ceramic solubility rate, the more pronounced bone ingrowth is
observed [25]. Ducheyne et al. hypothesized that the dissolution of Ca-P and precipitation of a car-
bonated HA layer is essential in the bioactive behavior of ceramics. On the other hand, Davies claimed
that microtopography played a crucial role as bone formation at the implant surface was achieved by
micromechanical interdigitation of the cement line with the material surface [26].
Protein–biomaterial interactions also play a key role in the biomaterial behavior in vivo. Directly after
implantation, the surface of a biomaterial is being covered by serum proteins, which determine crucial
reactions such as complement activation, thrombogenicity, and cell adhesion. Protein binding capacity
depends on the specific ceramic characteristics as well as on the protein involved [27,28]. Ceramic mater-
ials exhibit a high binding affinity for proteins. Interestingly, several authors report that the combination
of a material with high protein affinity and an appropriate (micro)architecture, can exhibit intrinsic
osteoinductive behavior [8,11,12]. In a study in dogs, Yuan et al. observed ectopic bone formation
after 3 and 6 months in micro- and macroporous HA, whereas macroporous HA without microporous
structure did not reveal bone formation. It was hypothesized that due to the microporosity, total sur-
face area and subsequent protein adsorption was increased, which could have induced bone formation.
On the other hand, Ripamonti et al. hypothesized that concave macroporosity was the driving force for
intrinsic osteoinduction in ceramics. The findings of intrinsic osteoinductive behavior in ceramic are
interesting. However, it must be emphasized that regarding the numerous studies using similar porous
ceramics without evidence for intrinsic osteoinduction, these observations currently are a rarity without
confirmation of reproducibility.
Protein adsorption on ceramics not only plays an important role in biological processes, it also influ-
ences the ceramic itself with respect to dissolution and precipitation of calcium and phosphate ions. Radin
et al. [29] reported that in an in vitro environment, serum proteins slowed down the dissolution (OHA
and β-TCP) and precipitation (carbonated HA) of crystals at the surface of a crystalline multiphasic
ceramic coating. A similar observation was done by Ong et al. [30], who reported an initial decrease in
dissolution of phosphate molecules from Ca-P coatings in the presence of proteins with a high binding
affinity. Combes et al. [31] investigated the interference of protein adsorption and HA maturation in
detail and found that, depending on the concentration, serum albumin can exhibit a dual role and act
as a promotor or inhibitor of nucleation and growth of (octa)Ca-P crystals. The energy needed for the
first step in the precipitation process, nucleation, was lower when adsorbing molecules were present.
Therefore, multiplication of nuclei occurred as a consequence of protein adsorption. The subsequent
steps, growth to critical nuclei and formation of Ca-P crystals, were slowed down by a protein coat-
ing. However, at low serum albumin concentrations, the mineral surface could not be efficiently coated
and crystal growth occurred faster than in the absence of albumin due to the larger number of nuclei
and their greater reactivity. At high — physiological — serum albumin concentration, the mineral sur-
face was efficiently coated slowing down the ionic diffusion to the crystal surface and inhibiting crystal
growth [31].
9.5 Calcium Phosphate Ceramics for Bone Tissue
Engineering
For reconstruction of large bone defects, implantation of osteoconductive porous scaffold materials
alone is not sufficient for complete bone regeneration. Therefore, osteogenesis needs to be induced
throughout the scaffold material. In the tissue engineering paradigm, osteogenic cells or osteoinductive
macromolecules or both are being delivered to a suitable scaffold material. Many prerequisites have
been postulated for the ideal scaffold material but none of the current materials meet all the demands.
Porous ceramics are potential scaffold materials for bone tissue engineering due to their outstanding
osteocompatible properties, especially with respect to osteoconduction and gradual resorption. In the
recent years, a variety of researchers have investigated Ca-P ceramic scaffold materials for cellular as well as
growth factor based tissue engineering.
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Calcium Phosphate Ceramics for Bone Tissue Engineering 9-9
Bone
OC
BM
HA/TCP
FIGURE 9.3 Bone formation in rat bone marrow stromal cell loaded porous HA/TCP implants (Camceram®),
implanted in muscular flaps in rats for 8 weeks. Direct apposition of bone on the ceramic surface and bone marrow
formation in the remaning porosity is observed. OC = osteocyte, BM = bone marrow. Bar represents 100 µm. Light
microscopical undecalcified section stained with methylene blue and basic fuchsin (original magnification 10×).
(Printed with permission from Hartman et al. Unpublished work, 2004.)
9.5.1 Ca-P Ceramics and Osteogenic Cells
In 1988, Maniatopoulos et al. published a study on harvesting and culturing of rat bone marrow stromal
cells (BMSCs) in vitro [32]. Through this method, the small number of undifferentiated progenitor cells
present in bone marrow could be isolated, expanded, and differentiated into the osteoblastic lineage.
Currently, the essence of this technique is still in use to administer osteoprogenitor cells to various scaffold
materials for cell-based bone engineering. Many studies in rodent animal studies have shown the validity of
this concept in conjunction with ceramic scaffold materials as coralline HA, porous HA, porous HA/β-TCP
and porous β-TCP [33–38] (Figure 9.3). Less studies, however, have investigated the bone forming
capacities of large BMSC loaded ceramic implants in higher mammals. This is in particular challenging,
as the living constructs are transposed from ideal cell culture conditions to an in vivo environment where
oxygen supply prior to capillary ingrowth is only provided by (limited) diffusion. Vitality of the BMSCs
within ceramic scaffold material was shown to be essential for osteogenicity [39]. Despite the potential
jeopardy of cell death, bone formation was observed in the studies investigating ectopic implants in dogs
and goats [40,41], or critical size defects in dogs, goats, and sheep [41–46]. In most of these studies,
bone formation was also observed in the center of the implants where the unloaded controls did not
show osteogenesis. This indicates that possibly even a small number of surviving progenitor cells may
be sufficient to result in significant bone formation. Also, it emphasizes that future tissue engineering
research should also focus on multicomponent constructs, in which BMSCs are being combined with
bone- and vasculature inducing growth factors.
9.5.2 Ca-P Ceramics and Osteoinductive Growth Factors
During the last decade, several strategies have been developed to deliver growth factors to the desired
site. Basically, the proteins can be delivered directly via a carrier/delivery vehicle, or by gene therapy
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9-10 Tissue Engineering
100
80
60
40
20
0
0 12243648
Hours
Normalized retention
FIGURE 9.4 Retention of
131
I rhBMP-2 injected subcutaneously in rats. Over 80% of rhBMP-2 was cleared from
the application site within 24 h, underlining the short residence time of rhBMP-2 after release from a carrier material.
Error bars represent means ± standard deviation for n = 6.
techniques, in which a growth factor encoding gene is delivered to host cells via a viral factor or plasmid.
Through this method, transfected cells start secreting growth factor. Cells can be transfected in vitro,prior
to transplantation to the desired site, or directly in vivo. In this review, only direct delivery of proteins
will be discussed as this approach has developed furthest and recently has led to FDA approval for specific
clinical applications [47].
The growth factor loaded ceramic has a twofold function; first, it serves as a scaffold material, which
enables and stimulates bone and blood vessel ingrowth; second, it serves as a carrier or delivery vehicle
for the inductive factor, which should result in most favorable biopresentation and optimal activity of the
protein. The role of ceramics on the bioactivity of growth factors is not completely clear. The ceramic
might potentiate the activity of BMP by binding the protein and presenting it to target cells in a “bound”
form [48,49]. On the other hand, the ceramic can act as vehicle for factor delivery to the surrounding
tissues. Released protein may provide a supra-physiological concentration of free protein in the vicinity
of the implant, what may attract target cells to the implant-site by chemotaxis [50]. It is hypothesized
that for optimal bioactivity, initial burst release chemotactically recruits osteoprogenitor cells, and fol-
lowing sustained release — or retained factor — differentiates these cells toward the desired phenotype
[51]. To our judgment, however, no convincing evidence provides further specification of this partition.
Moreover, optimal pharmacokinetics are presumably specific for each surgical site.
9.5.2.1 Growth Factor Release from Ca-P Ceramics
The correlation between factor bioactivity and release or retention is a delicate issue. Since the protein
needs to induce bone locally and not systemically, the released protein only has a short time to attract
cells before it is being cleared from the application site. For example, a depot of 5 µg rhBMP-2 injected
subcutaneously in the back in rats is cleared from the application site for more than 80% within 24 h
(Figure 9.4). After clearance, the proteins are being metabolized by the body and excreted through the
urinary tract [52]. The systemic exposure to released rhBMP-2 is reported to be neglectible [52].
The specific parameters resulting in protein release or retention are the driving factors behind the final
clinical efficacy of a carrier system. Unfortunately, only few researchers have investigated these phenomena
for ceramics. In a series of experiments in a rat ectopic model, Uludag et al. [53,54] have shown that growth
factor retention depend on carrier- as well as on factor characteristics. Proteins with a higher isoelectric
point (pI) show higher implant retention and higher retention of BMP yields higher osteoinductive
activity. Various rhBMP-2 loaded ceramic implants (TCP cylinders and coral derived HA cylinders) show
initial burst release of approximately 70% within three hours after implantation, which is followed by
a slower second phase release and a final retention of approximately 6% of the initial dose after 14 days.
In another study carried out by Louis-Ugbo et al. [52], initial burst release from HA/TCP granules (60 : 40)
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Calcium Phosphate Ceramics for Bone Tissue Engineering 9-11
was less than 20% within the first day in a rabbit spinal fusion model. After 2 weeks 27 ±9% of rhBMP-2
was still retained in this model. Li et al. [55] determined the release kinetics of rhBMP-2 mixed through
Ca-P cement (α-BSM) and injected in a rabbit ulnar defect. Within the first day, approximately 40% of
the initial factor was released. After 14 days, 15 ± 7% of the initial factor was still present in the cement.
The differences in release and retention rates between these studies could be explained by variations
in surgical site, dose, adsorption procedures, material surface area and chemico-physical interactions
between material and growth factor. Overall, ceramics exhibit multiphasic release kinetics in vivo with a
decrease of release in time and a certain retention for several weeks. It has been hypothesized, that ceramic
surface area plays the key role in protein binding [56].
As previously mentioned, Ca-P ceramic materials show a high binding affinity for proteins. Con-
formational changes of proteins upon adsorption on Ca-P surface have been observed [30]. Protein
conformation is of great importance for the accessibility of the specific binding sites and subsequent
protein-cell interaction [57,58]. As a result, protein-biomaterial bonding may directly effect the protein’s
intrinsic function due to orientation or conformational changes. To evaluate the bioactivity of retained
proteins, cells can be cultured on the surface of protein loaded ceramics in vitro [59]. A complicating factor
is that no material exhibits a 100% retention [54]. For a true differentiation between activity expressed
by retained protein vs. presently released protein, control groups have to correct for the released fraction
bioactivity. In an in vitro model, Santos et al. [59] investigated the bioactivity of rhBMP-2 adsorbed onto a
Ca-P layer on bioactive glass-gel (Xerogel S70) in comparison with the activity of “free” rhBMP-2 present
in cell culture medium. The osteogenic response of osteoblast like cells seeded on the Ca-P surface was
found significantly higher in the adsorbed rhBMP-2 group. The authors suggested that the biomaterial
could generate, maintain, or even concentrate an effective level of growth factor.
9.5.2.2 Growth Factor Loading in Ca-P Ceramics
In view of the previous paragraph, discern has to be made between proteins/growth factors adsorbed
at the ceramic surface and proteins/growth factors mixed through the material during preparation. For
sintered ceramic materials, the latter is impossible as sintering temperatures would result in complete
protein destruction. For the self setting Ca-P cements though, mixing of proteins during setting reaction
has been described [60–62]. Due to physical entrapment by cement particles, the majority of the protein
binding sites will be unavailable until the protein is released from the cement. This situation is quite similar
to bioactive macromolecules entrapped in polymeric drug delivery vehicles, where protein release is the
main focus [63–68]. However, a frequently neglected issue is that mixing of proteins through ceramics can
influence the crystallization process during the setting reaction. This can modify the in vivo dissolution
and/or degradation properties of the formed material.
For the growth factors entrapped within a Ca-P ceramic material, bioactivity is likewise expected only
after release from its carrier. Bioactivity of released protein can be demonstrated through in vitro models
where sensible cells are being exposed to the released protein [55,64,68]. To evaluate loss of activity, the
induced biological response should be compared to the response to similar protein directly suspended in
the culture medium [55]. In vivo, however, this comparison is difficult and most authors just examine
whether or not the protein expresses (part of its) bioactivity.
9.5.2.3 Osteoinductive Capacity of Growth Factor Loaded Ca-P Ceramics
Osteogenic differentiation induced by BMPs is dependent on the regulating growth factor as well as on the
carrier specifications. Murata et al. [69] observed that in a synthetic HA carrier, BMP induced ossification
was predominantly intramembranous, whereas enchondral bone formation was observed in a nonceramic
carrier. Direct bone formation on BMP loaded Ca-P ceramics was also observed by others [70–73].
However, the osteogenic behavior of growth factor loaded Ca-P ceramics is not as straightforward as
suggested, because a wide variety of material compositions, animal models, surgical sites and factor dosage
have been used in various studies. Another complicating factor is that physiological bone regeneration is
induced by a cascade of growth factors, whereas most tissue engineered concepts investigate osteoinduction
by single factors only. Consequently, most research in the recent years has focused on BMPs, like rhBMP-2
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9-12 Tissue Engineering
BV
OB
Bone
BM
Cement
FIGURE 9.5 Bone formation in rhBMP-2 loaded porous Ca-P cement implants, implanted subcutaneously in
rabbits for 10 weeks. Macroporosity in the cement (Calcibon®, Biomet Merck, Darmstadt, Germany) was induced by
formation of CO
2
during setting of the cement, after which a dosage of 5 µg rhBMP-2 was applied. OB = osteoblasts,
BM = bone marrow, BV = blood vessel. Bar represents 100 µm. Light microscopical undecalcified section stained
with methylene blue and basic fuchsin (original magnification 10×). (From Kroese-Deutman, H.C., Ruhe, P.Q.,
Spauwen, P.H., and Jansen, J.A. Biomaterials, in press: 2004. With permission.)
and rhBMP-7 (OP-1). Due to the numerous parameters, there is no such thing as one ideal dosage
or scaffold composition. Most animal studies investigating growth factor loaded ceramics have been
carried out in smaller animals as rats [9,10,53,54,69,70,72,74–78] and rabbits [52,55,73,79–85] (Figure 9.5)
whereas only few studies were performed in larger animals as minipigs [86,87], dogs [71,88,89], and
primates [90–92]. Generally speaking for all carrier materials, the relative dosage of growth factor needed
for bone induction raises with the animal size [51]. However, the dosages applied to ceramics vary
substantially within similar animal models as well as between different species. For rhBMP-2, dosages
applied for effective osteoinduction varied from 0.03 to 0.46 mg/ml implants in rats [9,54,75] to 0.004
to 2.1 mg/ml implant in rabbits [55,73,80,81,83,85], whereas in canine and primate models dosages have
been used from 0.15 to 1.2 mg/ml [71] and 1.35 to 2.7 mg/ml [92] respectively. A dose–response relation
for BMP loaded ceramics has been reported in some studies in rodents [75,80,83], but was absent or even
reciprocal in studies with larger animal models [71,91,92]. Yet, it seems likely that in the dose–response
relation, there is first a certain threshold dosage to achieve consistent results [92], followed by a linear
dose–response interval and finalized by a certain saturation effect.
Besides BMPs, other growth factors like transforming growth factor-β (TGF-β) and insulin-like growth
factor-I (IGF-I) have been investigated. TGF-β may potentiate the osteoinductivity of BMPs, but its
overall osteoinductive capacity when applied alone is weak compared to BMPs [93]. IGF-I has also been
applied to TCP ceramic carriers, which resulted in increased osteogenesis in an orthotopic implantation
site four weeks after implantation [94]. Similarly, Damien et al. [95] reported that IGF-I loaded porous
hydroxyapatite accelerated orthotopic bone ingrowth during an implantation period up to three weeks.
The synergism of various growth factors, as present in the physiological bone regeneration cascade, has
only been investigated in few studies with Ca-P ceramics. Ono et al. investigated the role of prostaglandin
E
1
(PGE
1
) and basic fibroblast growth factor (bFGF) loaded on porous hydroxyapatite and observed
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Calcium Phosphate Ceramics for Bone Tissue Engineering 9-13
that PGE
1
as well as bFGF acted synergistically with rhBMP-2 [80,83,96]. Meraw et al. prepared a true
growth factor cocktail and reported that orthotopic bone formation in a canine model was enhanced by
a combination of extremely low dosages of TGF-β, bFGF, BMP-2, and PDGF mixed through a calcium
phosphate cement [89].
9.6 Conclusion and Future Perspective
Like all scaffolds materials currently under investigation for bone tissue engineering applications, Ca-P
ceramics reveal both advantages and disadvantages. Intrinsic brittleness of Ca-P ceramics unquestionably
is the greatest shortcoming of these materials, whereas their biocompatibility, osteoconductivity and
resorption potential are virtues of an ideal scaffold material. Future research should further clarify the
importance of release and retention of growth factors, optimize their delivery and investigate dose–
response relation for the various factors and applications. Furthermore, the potentials of multicomponent
constructs, in which various bone- and vasculature inducing growth factors or cultured bone marrow
stromal cells or both are being combined, should be investigated to come to a successful alternative for
autologous bone grafting.
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