The development of injectable and self-hardening biomaterials able to regenerate damaged bones by minimal invasive surgery is a topic of increasing interest, particularly for the treatment of vertebral fractures [1
], but also tibial plateau, proximal humerus, wrist and calcaneus [6
], as well as hip and femoral neck [7
In this respect, complete bone regeneration still remains an unsolved clinical need, mainly due to the unavailability of bone cements endowed with adequate bioactivity, porosity, and osteoconductivity able to promote effective new bone formation and extensive cell proliferation upon hardening. Indeed, it has been reported that the main drawback exhibited by commercially available bone cements is the inability to be completely colonized by the new bone, thus affecting the establishment of well-integrated bone-biomaterial constructs [8
]. The impairment of the mechanical performance may be due to both the excessive stiffness of the cement, as reported in the case of acrylic formulations [11
], and the low porosity, as reported in the case of calcium phosphate bone cements (CPCs) [1
The hardening of acrylic bone cements (e.g., poly(methylmethacrylate), PMMA) is also reported to occur through an exothermic polymerization, leading to the necrosis of the surrounding tissues [16
]. In this respect, CPCs are considered as elective candidates for bone regeneration due to their excellent biocompatibility, chemical similarity with bone tissue, and ability to harden in vivo at body temperature.
Several approaches have been proposed to prepare CPCs; among them, the one based on the hydrolysis and transformation of α
TCP) into elongated calcium-deficient hydroxyapatite (HA) particles is particularly interesting and promising [1
]. Indeed, the chemical reactions involved in the cement hardening are isothermal and not subjected to pH variations, thus avoiding potential cell injury. Calcium-deficient HA is also characterized by increased bioactivity and bio-resorbability, if compared with stoichiometric HA [17
]. Moreover, such cements can be endowed with enhanced bioactivity by introduction of bio-competent ions (e.g., Mg2+
, and Sr2+
) in the HA lattice as substitutes of calcium or phosphate [18
]. Strontium has been largely studied due to its proven ability to restore the bone turnover balance, especially when the treatment of bone fractures caused by osteoporosis is demanded [22
]. As systemic pharmacological approaches based on Sr-containing drugs often resulted in deleterious effects on bone mineralization due to reduction in calcium absorption and possible alterations of the properties of the mineral bone [24
], a controlled delivery of relevant active principles in situ is highly desirable to provide safe and efficient therapies against degenerative bone diseases. Some positive results on the efficacy of local release of strontium were obtained in recent studies based on titanium implants functionalized with strontium [25
] or coated with Sr-substituted HA [26
]. In addition, Sr-containing CPCs based on commercial α
TCP and soluble salts containing strontium (e.g., SrCO3
) were previously developed [27
] and tested in ovariectomized rats [30
A major challenge for material scientists is the control of the chemistry and rheological properties of injectable CPCs; indeed, process parameters, such as phase composition of the inorganic precursor, particle size, liquid-on-powder ratio (L/P), polymeric additives are critical factors affecting their viscosity, cohesion, and setting times [15
]. In this respect, previous research reported improved cement injectability and cohesion by decreasing the particle size of the starting powders, as well as using more viscous solutions. However, in such conditions the increase of the cement cohesion was also associated to an impairment of setting times and mechanical performance [31
]. It was also reported that smaller precursor particles favor the transformation into apatite, as well as the establishment of a more packed structure and higher mechanical strength [32
]. On the other hand, the introduction of foreign ions into the calcium phosphate structure prolonged the setting times [29
]. Among the various approaches proposed to improve the hydraulic reactivity of the precursor powders, the high-energy planetary ball milling was reported to be particularly promising in terms of optimal particle size reduction efficiency [36
]. Previous research was also dedicated to improve the rheological properties of the cement by addition of organic compounds such as biopolymers [15
]. In this respect, alginate was reported as a biomaterial for bone tissue engineering, due to its biocompatibility, non-toxicity, and biodegradability, as well as for its strengthening ability [37
In the present work Sr-doped HA cements were prepared by mixing Sr-substituted αTCP phases, appositely designed and synthesized to be the unique inorganic precursors, with disodium phosphate solutions enriched with alginate. The process parameters, e.g., the powder milling and liquid-to-solid ratio, were optimized to obtain adequate setting times and mechanical properties. The in vivo performance of a selected cement formulation was preliminary evaluated at four weeks after surgery into rabbit femur, in comparison with a commercial CPC, by compositional, morphological, and histological/histomorphometric analysis.
In the present work, Sr-substituted α
TCP phases were synthesized as a unique inorganic precursor for the development of apatitic bone cements. For the first time, Sr-substituted apatitic cements were obtained by incorporating sodium alginate and tested both in vitro and in a small in vivo pilot study. The cements were optimized to fulfill specific requirements for clinical applications in terms of hardening times and mechanical strength [42
The precursor synthesis, based on solid state reactions followed by fast quenching, generated Sr-doped α
TCP phases with limited amounts of β
TCP polymorph, slightly increasing with the strontium content, as previously reported [3
]. This results are in compliance with the larger ionic radius of strontium compared with calcium (Sr2+
ionic radius = 0.113 nm; Ca2+
ionic radius = 0.099 nm) and in good quantitative agreement with the data provided in previous works [38
]. In this respect, the detection of Sr amounts very close to the nominal composition of the starting mixture (Table 1
), associated with the absence of secondary phases other than TCP (Figure 1
) and the simultaneous increase of the lattice parameters (Figure 2
), confirmed the incorporation of strontium into the TCP crystal lattices and in the final HA phase obtained upon cement setting.
The achievement of complete injectability, associated with adequate setting times, is reported to be one of the most critical issues of CPCs [8
]; in this respect, the addition of polymeric additives into CPCs has been widely investigated in previous research to enhance the rheological properties, but the precise control of their effect still remains challenging [44
In our cements, characterized by ionic substitutions with strontium and the presence of alginate, a significant retard in both setting times (Table 3
), and the transformation of TCP phases into apatite (Figure 4
) were detected. However, this effect can be mainly ascribed to the presence of strontium, as also hypothesized in previous works [3
], whereas the effect of alginate appeared as negligible.
As a general rule, comparative analyses among the various cements reported in literature are made quite difficult by the variety of different, and often incomparable, preparation routes [20
], thus often leading to contradicting results.
It was reported that the incorporation of biopolymers can improve the functional properties of CPCs [44
]. In this respect, despite previous research reporting the addition of alginate as deleterious for the mechanical performance of α
TCP-based CPCs formulations [46
], in the present work the use of alginate significantly improved both injectability and cohesion (Figure 3
), leading also to significantly higher mechanical strength when compared with alginate-free cements (Figure 6
). The effect of strontium on the mechanical strength appeared as negligible in alginate-free cements up to Sr2, while Sr5 exhibited the lowest values; a similar trend, but with greater statistical difference, was also detected among the alginate-containing cements, with Sr2 exhibiting the highest compressive strength (Figure 7
), even comparable to previously-reported CPCs reinforced with fibers [15
The reinforcement mechanism provided by alginate could be due to a crack bridging effect, similarly to what proposed by [47
] in the case of cellulose-containing CPCs.
On the other hand, a previous study reported a decrease of the mechanical strength of Sr-CPCs above a certain threshold of strontium [48
], possibly due to the higher occurrence of crystal lattice distortion related to strontium’s larger ionic radius; in our study, we observed a similar effect for Sr/(Sr + Ca) above 2 mol %.
For the first time the biological effect of strontium doping and alginate into an apatitic cement was tested with MG63 cells, resulting in excellent proliferation and spreading of the seeded cells (Figure 8
). This finding confirms previous studies reporting beneficial effects of strontium on cell proliferation in vitro [20
], while the absence of cytotoxic effects related to alginate was assessed, supporting its use in the development of biomaterials for bone regeneration.
On the basis of adequate cement composition and setting time for clinical applications [42
], the Sr2 cement was selected to be mechanically and in vivo tested in comparison with KyphOs, a commercial strontium-containing apatitic cement [8
] as control. After one month in vivo the Sr2 cement showed significantly higher bone-material contact and bone penetration (Figure 9
and Figure 10
), perhaps due to the higher porosity. It might be also hypothesized that the presence of alginate favored the penetration of bone tissue into the cement by progressive bio-erosion in vivo. Comparative in vivo tests should be performed to support this hypothesis; however, in the present work we found that alginate-free pastes did not reach acceptable rheological and mechanical properties, as well as adequate setting times when injected in liquid media, so as to be considered as not interesting for real applications.
The XRD analysis of the explanted tissues reported that the composition of the control cement did not change significantly after one month in vivo, with only a partial conversion of the precursors into hydroxyapatite. Conversely, Sr2 exhibited complete transformation into hydroxyapatite in vivo, thus leading to the establishment of a completely biomimetic composition (Figure 11
). In Sr2, the analysis of the HA peak profile evidenced a marked reduction of the crystal domain size. In this respect, it can be hypothesized that Sr2 underwent also a degradation process following the new bone formation and penetration.
The SEM analysis of the explants reported the maintenance of an extensive microporosity in Sr2 even after one month in vivo, while a more packed structure was exhibited by the control cement, that might have limited bone penetration into the cement mass (Figure 12
). KyphOs exhibited significantly higher compressive strength but also lower overall porosity (Figure 7
). Porosity substantially influences both the biological activity and the mechanical properties of biomaterials, therefore their optimization is a key aspect in the development of a bone cement. The microporosity of CPCs, which usually varies between 30% and 55%, is significantly dependent on the L/P ratio: the higher the L/P ratio, the higher the microporosity [15
]. Therefore, to optimize these two properties, L/P ratio was maintained at the lowest value enabling complete injection and adequate cohesion. The final L/P of Sr2 resulted slightly higher than the control cement, i.e., 0.48 and 0.43, respectively; this yielded a much higher porosity associated with nanosized elongated crystals, as typically occurs in apatitic CPCs [3
], which might have favored osteointegration.
The bioactivity and effective osteointegration of a bone scaffold are key aspects to trigger and sustain bone regeneration in critical size defects [51
]. In this respect, the approach presented in this work enabled the preparation of Sr-substituted HA cements with controlled and biomimetic composition, with defined strontium content replacing calcium in the lattice of HA, associated to excellent injectability, cohesion, and stable compressive strength enabling mechanical loading. The new cement maintained also high bioactivity and porosity in vivo, making these new pastes interesting for future applicative purposes.
Although other cement formulations (e.g., PMMA-based cements) enable the early-stage mobilization of the patient after surgery due to high mechanical strength, their intrinsic bioinertness prevent effective bone regeneration. In this respect, the implantation of bioactive apatitic pastes able to completely transform into apatite while maintaining a microporous structure can be elective for bone regeneration, due to the extensive colonization by new bone that can also improve the mechanical performance of the implant over time, as already evidenced in previous studies [52
]. This is particularly relevant in the case of young and still physically active patients, for whom effective bone regeneration is of paramount importance to restore the physiological bone functionality.