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Tissue Engineering - John P. Fisher

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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 carbonated 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 materials 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 microand macroporous HA, whereas macroporous HA without microporous structure did not reveal bone formation. It was hypothesized that due to the microporosity, total surface 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 influences 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 coating. 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 surface was efficiently coated slowing down the ionic diffusion to the crystal surface and inhibiting crystal growth [31].

9.5Calcium 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.

Calcium Phosphate Ceramics for Bone Tissue Engineering






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 boneand 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


Tissue Engineering

Normalized retention













FIGURE 9.4 Retention of 131I 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 following 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. 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 carrieras 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)

Calcium Phosphate Ceramics for Bone Tissue Engineering


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. Conformational 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. 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. 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


Tissue Engineering






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 CO2 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 E1 (PGE1) and basic fibroblast growth factor (bFGF) loaded on porous hydroxyapatite and observed

Calcium Phosphate Ceramics for Bone Tissue Engineering


that PGE1 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 boneand 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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