Fig. 4.1
3D reconstruction images of residual β-CS and β-TCP after implantation in the rabbit calvarial defects for different periods using Micro-CT analysis (From Xu et al. [219], with permission)
The pseudowollastonite (α-CaSiO3), which is a high temperature form of calcium silicate, has also been found exhibiting good biocompatibility and bioactivity. Dufrane et al. [220] showed that the pseudowollastonite extract did not show significant cytotoxic effects confirming its biocompatibility. Lin et al. [221] also demonstrated good biocompatibility of α-CaSiO3. The bioactivity of pseudowollastonite has been observed in vitro (in SBF) and in vivo (implanted in animals). Apatite formation on the surface of α-CaSiO3 scaffold after soaking in SBF is shown in Fig. 4.2. Similar to wollastonite, pseudowollastonite also has the ability to induce apatite formation when immersed in SBF [197]. It can even induce apatite formation in human parotid saliva [195] and serum-containing media [10]. It has been reported that the rate of hydroxyaptite precipitation on the surface of pseudowollastonite surface are higher than those on all the reported bioglasses and glass-ceramics [222]. Sarmento et al. [198] found that osteoblasts could attach and proliferate well on the surface of pseudowollastonite. In addition, the cell attachment could be enhanced by preincubation of pseudowollastonite in serum or media containing fibronectin. The in vivo bioacticity of pseudowollastonite was evaluated by De Aza and co-workers through implantation into rat tibias [196, 199]. The SEM and EDS analyses showed that a calcium phosphate layer was formed at the implant interface, which had characteristics of new bone tissue. High resolution transmission electron microscopy observations confirmed the newly formed bone at the interface between the pseudowollastonite implant and the host bone as composed of hydroxyapatite-like nanocrystals growing epitaxially across the interface in the [002] direction [196]. It was shown that the rate of new bone formation around pseudowollastonite decreased after the first 3 weeks and reached constant value over the following 9 weeks, which coincided with the results of β-wollastonite reported by Xu and co-workers [219].
Fig. 4.2
Apatite formation on the surface of α-CaSiO3 scaffold after soaking in SBF (From Lin et al. [221], with permission)
Sahai et al. [223] used crystallographic constraints with ab initio molecular orbital calculations to identify the active site and reaction mechanism for heterogeneous nucleation of calcium phosphate. It is proposed that the cyclic silicate trimer is the universal active site for heterogeneous, stereochemically promoted nucleation on silicate-based bioactive ceramics. A critical active site density and a less point of zero charge of the biomaterial than physiological pH are considered essential for bioactivity.
Chang and his colleagues find that dicalcium silicate and tricalcium silicate also show good bioactivity, and they can rapidly induce apatite formation in the SBF [201–203, 224]. Besides the binary calcium-silicates, some ternary calcium-silicate ceramics have also been attracted much attention in recent years. The investigation of diopside as implant material started by Nakajima in the late 1980s [225]. It was found that diopside can induce apatite formation in SBF and can form a bone bonding with surrounding bone tissues [209, 226, 227]. Calcium released from the material into SBF plays a key role in the apatite formation on the surface of diopside, which is initially released rapidly and eventually reaching steady-state. On the contrary, Mg and Si are released more slowly at similar rates to each other [12, 211, 228]. And Mg does not play a role for apatite nucleation on diopside [211]. It is proposed that the (100) plane of diopside epitaxially nucleates the (010) plane of octacalcium (OCP), which has a similar cell parameters to hydroxyapatite and has been considered as a precursor to hydroxyaptite in normal bone growth [12, 227]. The reported bending strength and fracture toughness of the diopside is 300 MPa and 3.5 MPa · m1/2, respectively [209]. These values are about two or three times higher than those of hydroxyapatite. However, the degradation rate of diopside is very poor, which is even lower than that of hydroxyaptite [209].
Besides diopside ceramics, akermanite and bredigite in the Ca-Si-Mg system have also been investigated for bone tissue engineering. Wu et al. [206, 229] synthesized pure akermanite and bredigite powders by sol–gel methods. Both akermanite and bredigite have the ability to induce apatite formation in SBF. The apatite formation ability decreases with the increase of Mg in the Ca-Si-Mg ceramics, i.e. bredigite has better apatite formation ability than akermanite, which is indicated by higher calcium content and lower phosphorus content in SBF after immersion. The increase in activation energy of Si release should be responsible for the reduced apatite formation ability [230]. In addition, activation energy of Si release also predominates the degradation rate of the Ca-Si-Mg ceramics. With the increase in Mg content, the degradation rate of the Ca-Si-Mg ceramics decreases. Considering the poor degradability of diopside, it may not be suitable as bone tissue engineering materials as the akermanite and bredigite. Akermanite prepared by two-step precipitation method has a higher bioactivity than that prepared by sol–gel method, due to its finer particle size. Akermanite and bredigite have all shown the ability to stimulate osteoblasts proliferation. The intensive investigation by Sun et al. showed that akermanite ceramics enhanced the expression of osteoblast-related genes, including alkaline phosphate (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OC) [231]. It also showed that akermanite could promote osteoblastic differentiation of human bone marrow stromal cells (hBMSC) in normal growth medium without osteogenic reagents, such as L-ascorbic acid, glycerophosphate and dexamethasone (as shown in Fig. 4.3). Highly connective porous akermanite and bredigite scaffolds, with porosity about 90 % and pore size ranging 300–500 μm, were prepared using polymer sponge as templates by Wu and his co-workers [208]. Both alkermanite and bredigite scaffolds could support osteoclasts-like cells growth, proliferation and differentiation. The biomimetic treatment of alkermanite and bredigite scaffolds in the SBF could enhance the cell proliferation and differentiation.
Fig. 4.3
ALP staining of differentiating hBMSC on the surface of different material. The hBMSC were cultured on akermanite disks (a, b) and β-TCP disks (c, d) for 7 days in growth medium (a, c) or osteogenic medium (b, d). ALP-positive cells are shown in purple. The bars in the pictures present 200 mm (From Sun et al. [231] with permission)
To combine the advantages of phosphates and silicates, Ning and her co-workers synthesized pure Ca5(PO4)2SiO4 (CPS) by a sol–gel method using triethyl phosphate (TEP), tetraethoxysilane (TEOS) and calcium nitrate tetrahydrate as original materials [213]. It is revealed that CPS has a greater in vitro apatite-forming ability than HA. In addition, the proliferation of rBMSC on CPS is significantly higher than that on HA. Moreover, the expression of alkaline phosphatase activity (ALP) and osteogenic-related genes, including Runx-2, osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), demonstrated that CPS has enhanced the osteogenic differentiation of rBMSC and accelerated the differentiation process [214].
Silicate/Phosphate Based Composites
As stated above, silicate-based bioceramics exhibit excellent bioactivity, which can promote the osteoblast proliferation, induce the osteoblastic differentiation of marrow stem cells, and enhance the bone formation. On the other hand, calcium phosphate ceramics have excellent biocompatibility due to their similar compositions to the bone minerals, while they have no obvious stimulatory effect on the proliferation and differentiation of osteoblasts. A composite strategy is applied to combine the advantages of silicates and phosphates, which is effective way to make materials with tailorable properties, such as mechanical property, bioactivity and biodegradation rate.
De Aza et al. [232, 233] developed a bioeutectic wollastonite-tricalcium phosphate ceramic, with a composition of 60 wt% wollastonite and 40 wt% TCP, by a specific high temperature treatment (termed W-TCP). The eutectic W-TCP material presented a high bioactivity in SBF [232] and human parotid saliva [233], with the formation of two well-differentiated zones of hydroxyapatite. The inner layer formed by pseudomorphic transformation of the tricalcium phosphate into hydroxyapatite after the dissolution of wollastonite into SBF, and the outer layer formed by the deposition of hydroxyapatite onto the surface of the material in the later stages of immersion.
Huang et al. [234] and Ni et al. [235] prepared β-CaSiO3/β-Ca3(PO4)2 composite materials by in-situ precipitation method. The mechanical properties of the CS-TCP composites increased with the increase in TCP content. A higher CS content also resulted in a higher dissolution rate. The CS-TCP composites exhibited good bioactivity. Compared with pure β-TCP, the CS-TCP composites, especially the composites with over 50 % wollastonite, enhanced the adhesion, growth and ALP activity of the osteoblast-like cells [235]. Zhang et al. [193] prepared nanocrystalline wollastonite/β-TCP composite powders by a two-step chemical precipitation method. Porous scaffolds were fabricated using these composite powders by porogen burnout technique. The mechanical properties of these scaffolds sintered from nano-scale composite powder were significantly improved, which were about twice as high as those of the scaffolds sintered from submicron powders. In addition, these scaffolds sintered from nano-powders showed less strength loss during the degradation process.
The silicate/phosphate composite ceramic with the composition of 32.9 mol% Na2O, 32.9 mol% SiO2, 22.8 mol% CaO and 11.4 mol% P2O5 were prepared by El-Ghannam and his co-workers [216], which showed main crystalline phases of Na2CaSiO4 and NaCaPO4 (termed SCPC). This composite ceramic has compositional components similar to 45S5 bioglass. SCPC provided a superior release profile of biologically active rhBMP-2 compared to commercial porous hydroxyapatite. Moreover, cells attached to the SCPC produced mineralized extracellular matrix and bone-like tissue covered the entire material surface after 3 weeks culture in vitro, while the hydroxyapatite only produced limited amount of unmineralized ECM. Porous SCPC scaffold was prepared by rapid prototyping technique using a segment of a rabbit ulnar bone as prototype model [215]. After 4-weeks, CT scans showed that the defect filled by the above SCPC composite scaffold loaded with rh-BMP-2 had already been replaced by newly formed bone, indicating that SCPC are highly resorbable and have good bone formation ability.
Polymer/Inorganic Composites
The composite materials for bone tissue engineering have been pursued in the near decade, since the composites combine the advantages of the different components, which offered superiorities over single-phase materials.
Compared to the strengths of metals and ceramics, the strengths of biodegradable polymers are low. The porous structure of the scaffolds further decreases their strengths. Moreover, the synthetic polyesters are often non-osteoconductive. To enhance the strength and bioactivity of the polymer scaffolds, an inorganic component is always introduced to make polymer/inorganic composites. Studies have demonstrated that such composites could result in scaffolds with tailorable physical and biological properties for specific applications. The addition of an inorganic phase to a biodegradable polymer may also change the in vitro and in vivo polymer degradation behaviour.
Bioglass, glass-ceramics, calcium phosphates and silicates, etc. have all been used to reinforce polymers. The development of polymer/inorganic composites has been well reviewed in literatures [4, 13, 32]. In the recent years, the polymer/silicate ceramic composites have been intensively investigated. For example, wollastonite was incorporated into the PDLLA to prepare a bioactive PDLLA/wollastonite composite [236]. The composite scaffold was prepared using a solvent casting/particulate leaching method. With the same salt content, the porosity of the PDLLA deceased from 95 to 85 % as the wollastonite content increased from 0 to 40 %. The bioactivity of the PDLLA/wollastonite composite was confirmed by the formation of an apatite layer on its surface after immersing in SBF for seven days. The interesting and important advantage of the PDLLA/wollastonite composite is that the acidic degradation products of the PDLLA could be neutralized by the basic ions released from wollastonite due to its dissolution in the SBF solution. For the PHBV/wollastonite porous scaffolds, there were no significant differences in porosity between the samples with different wollastonite content [237]. However, the mechanical strength of the composite scaffolds was significantly enhanced by the incorporation of wollastonite. In addition, the incorporation of silicates into polymers will result in an improvement in hydrophilicity, expressed by a decrease in water contact angle [237, 238]. This implied that wollastonite could be used as a good candidate for preparation of bioactive polymer/ceramic composites for tissue engineering applications.
During the process of polymer/ceramic composites preparation, a common problematic issue is that it is difficult to get a uniform polymer/inorganic particle suspension, since the inorganic particles have the tendency to agglomerate. This problem makes it difficult to fabricate composites with a uniform microstructure [236, 237]. And it was found that some of the PDLLA/β-CaSiO3 composites lost their strength rapidly under physiological environment, and failures mainly occurred at the interface between the β-CaSiO3 agglomerates and the polymer matrix. Consequently, it is necessary to increase the compatibility between the inorganic component and the polymer matrix by improving the dispersion of inorganic particles in preparing polymer/inorganic composites.
Mechanical stirring [239] and ultrasonic energy [240] have been used to reduce agglomerate formation and provide some level of particle dispersion during the blend processing of composite. However, these effects are just temporary and particle agglomeration ensues once the mixing energy is removed.
It is supposed that chemical techniques can provide more permanent effect to solve this problem and various methods have been developed to match the surface properties between filler powders and a specific polymeric matrix [241–244]. Zhang et al. [241] used silane derivatives as modification molecules to shield hydroxyl groups (−OH) formed on the surface of HA to improve the interfacial property between the ceramic phase and the polymer phase, which resulted in a 27.8 % increase in maximum bending strength of the HA/PLA composites. Qiu et al. [242] modified the surface of HA with L-lactic acid oligomer, and the dispersion of HA particles in the polymer solution was improved significantly. The mechanical strength of the L-lactic modified HA/PLLA composite film was also increased [242, 243].β-CaSiO3 particles treated with dodecyl alcohol can react with the Si-OH groups on the surface of β-CaSiO3 particles in an aqueous solution by esterification reaction. This modification could make the β-CaSiO3 particle hydrophobic and thus enhance its dispersion in the organic solvent (as shown in Fig. 4.4). The tensile strength of the modified β-CaSiO3/PLLA composite film with 15 wt% ceramic phase increased 52.2 % compared to that of the unmodified one [244]. In addition, the modification had no effects on the bioactivity of the β-CaSiO3/PLLA composite. Our experiments also showed that the dodecyl alcohol on the modified CaSiO3 particles in the composite could be removed by hydrolysis in boiling water. The valuable results are that the esterification-hydrolysis process has improved the mechanical properties of β-CaSiO3/PLLA composites, while without impairing their wettability and bioactivity. The same phenomenon was found for the 45S5/PLLA composites.
Fig. 4.4
SEM micrographs of the composite films. (a) composite film with 15wt%β-CaSiO3 and (b) composite film with 15 wt% modified β-CaSiO3 (From Ye et al. [244], with permission)
Concluding Remarks
The concept of replacement of tissues has been shifting to a new concept of regeneration of tissues in the new century [142]. Tissue engineering is an effective way to achieve the goal of tissue regeneration. From the perspective of materials science, the present challenge in tissue engineering is to develop bioactive and bioresorbable biomaterials, which should have the ability to activate the body’s own repair mechanisms. An ideal biomaterial for bone tissue engineering should have favorite composition and structures which can facilitate cellular attachment, proliferation and stimulate osteoblastic differentiation of bone marrow stromal cells, and should initiatively participate in the activities of bone formation.
Generally speaking, silicate ceramics have superior bioactivity than phosphate ceramics. The former are considered as osteoconductive and may be considered as osteoinductive, while the latter are only considered as osteoconductive. Therefore, silicate ceramics have a more wide application perspective for bone tissue engineering than phosphate ceramics.
On the other hand, since the hard tissues in human body are natural composite materials, the composite strategy provides an effective way to fabricate scaffold biomaterial with tailorable physiochemical and/or mechanical properties. The composite scaffolds possessing both osteoconductivity and osteoinductivity appear to have great potential for bone tissue engineering applications.
In addition, the architecture of the scaffolds not only influences its mechanical properties and degradation behavior, but also strongly affects the cellular activities and nutrition supplies in the scaffold, which are also important factors for bone regeneration. Thus, the ideal scaffold material should also have highly connective porous structure.
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