Bisphosphosphonate-calcium phosphate cement composite and its properties

1. Introduction

Calcium phosphate cement (CPC) has been used in bone reconstruction extensively. In addition to bone applications [1], CPCs have been used in dental applications. Calcium phosphate materials were first used in the 1920s as bone grafts [2]. In a bony defect, a triple calcium phosphate compound was used to promote osteogenesis. In 1971, Monroe et al. reported a method for preparing calcium phosphate, and they suggested the use of this material for dental and bone implant applications [3]. Applications of CPCs include sinus lifts, tooth replacement, repair of periodontal defects, and alveolar bone augmentation [4]. CPCs may also be used in tissue engineering as a scaffold for dentin or bone regeneration [5]. Other potential dental applications of CPCs include cavity lining and pulp capping [6].

Despite a high quantities of proposed CPC formulations over the past 25 years, only two possible end products have come to fruition: brushite (viz. dicalcium phosphate dihydrate; DCPD, CaHPO₄⋅2H₂O) and apatites, including hydroxyapatite (HA; Ca₁₀(PO₄)₆(OH)₂) and calcium-deficient hydroxyapatite (CDHA; Ca₉(HPO₄)(PO₄)₅OH). Thus, CPCs are classified as either brushite CPCs or apatite CPCs [7]. In the early 1980s, the potential of obtaining a monolithic hydroxyapatite at bodily temperatures through a cementitious reaction was reported. This was a revolutionary development in the field of bioceramics for bone regeneration, as it offered a material that was not only bioactive and moldable but also had the capacity to self-set in vivo within the bone cavity. When dealing with drug delivery, a prospective matrix must be able to assimilate a drug, secure it in a specific target location and release it to the surrounding tissues over time. Additionally, biodegradability of the material and the ability to be injected are very beneficial [8]. Based on these characteristics, CPC is an ideal carrier of drugs to bone regions. CPC permits the usage of drugs in either liquid or powder phase, allowing the modification of CPC properties, namely hardening time, mechanical properties and others.

Another type of material that is used in bone treatment is bisphosphonate, which is used commonly for osteoporosis prevention and treatment. The name, bisphosphonate, is derived from its two phosphonate (PO(OH)₂) groups. Research has shown bisphosphonates to reduce the risk of fracture in post-menopausal osteoporotic women. They are divided into two groups based on whether or not they contain nitrogen. etidronate, clodronate and tiludronate are examples of non-nitrogenic bisphosphonates, while pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, incadronate, and zoledronate belong to the nitrogen-containing category. The key difference between the two groups is that bisphosphonates without nitrogen are swiftly metabolized; whereas, nitrogen-containing ones are much more powerful and are not metabolized easily [9]. The presence of a nitrogen group in a bisphosphonate increases its antiresorptive potency by 10 to 10,000 in comparison to non-nitrogenic bisphosphonates [10]. The mechanism by which nitrogenic and non-nitrogenic bisphosphonates promote osteoclast apoptosis is different. Nitrogenic bisphosphonates inhibit farnesyl pyrophosphate synthase (FPPS) in osteoclasts. FPPS is a key enzyme in the mevalonic acid pathway, which is necessary for the production of isoprenoid lipids and cholesterol. Thus, this leads to modification of proteins that are necessary for the regulation of osteoclast cellular activities, which leads to osteoclast apoptosis. Even though FPPS is expressed in all mammalian cells, cellular apoptosis caused by nitrogenic bisphosphonates only occurs in osteoclasts, likely due to the fact that bisphosphates selectively adhere to bone. In contrast, non-nitrogenic bisphosphonates are close in structure to inorganic pyrophosphate (PPi); thus, they become incorporated into new molecules of adenosine triphosphate (ATP). Accumulation of these nonhydrolyzable ATP analogues might be cytotoxic to osteoclasts and leads to osteoclast apoptosis. Currently, the more potent nitrogenic bisphosphonates are more commonly used in clinical applications as opposed to non-nitrogenic bisphosphonates.

So far, a bisphosphonate-calcium phosphate composite as a drug delivery system consists of any form of calcium phosphate cement with etidronate, alendronate, pamidronate, and zoledronate. There is no calcium phosphate cement composite that consists of clodronate, tiludronate, neridronate, olpadronate, ibandronate, or risedronate.

2. Alendronate

Jindong et al. created powdered calcium phosphate cement with alendronate (ALN) in 3 different concentrations (20, 50, and 100 mg/g), and discovered through Fourier transform infrared spectroscopy that the samples with and without ALN were nearly the same. Additionally, there was no noticeable difference in the compressive strengths of the samples; however, the use of ALN in the cement prolonged the setting time of the composite, increasing the time as the concentration of ALN increased. In 21 days, the released percentage of 2% ALN, 5% ALN, and 10% ALN were 33.2%, 24.4%, and 20.8%, respectively. Also, the high-ALN-content group had a significantly greater early release rate in comparison to the low-content group. Cytotoxicity after seven days showed that all groups had the same number of viable cells [11]. Panzavolta et al. [12] introduced gelatin in the preparation of ALN loaded cement. 𝛼-TCP (tri calcium phosphate) was mixed with various weight percentages (10, 15 and 20) of gelatin solutions, and 0, 12, 25 and 50 mM disodium ALN was added. With the addition of gelatin they could increase the amount of ALN incorporated into the cement (25 mM) and the setting times increased with increasing concentrations of the bisphosphonate with all percentages of gelatin. The compressive strength decreased as the ALN concentration increased with all percentages of gelatin. Gelatin addition successfully resulted in the transformation of 𝛼-TCP to CDHA after 14 days of soaking in stimulated body fluid (SBF) at a gelatin concentration of 20%. Osteoclast proliferation and differentiation were reduced significantly with the addition of ALN, while osteoblast proliferation was achieved. Verron et al. [13] incorporated ALN combined CPC in the proximal femurs of sheep presenting induced osteopenia for 12 weeks. The treatment showed substantial increase in bone volume/total volume percentages (BV/TV), trabecular thickness (TbTh) and trabecular number (TbN) values. They also observed micro-architecture and bone density modifications in the sheep injected with ALN-CPC.

In another study, Shen et al. [14] showed that both compressive strength and compactness increased with ALN loaded CPC. Injectability and setting time increased alongside increasing bisphosphonate concentration with 1% ALN concentration samples exhibiting more than 120 min of setting time. Initial and final setting times increased as the ALN concentration increased up to 2%, but decreased for 3% and more so for the 4% concentration. Though there was a burst release for all the groups, in vitro release percentage was highest for the 1% ALN group (91.3% ± 3.9%) after 19 days. ALN concentration of 1 wt.% exhibited the highest compressive strength of 31.62 ± 2.37 MPa, while this characteristic decreased with increasing bisphosphonate concentration. The compressive strength increased as the ALN concentration increased up to 1%, but decreased for 2% and more so for the 3% concentration.

Work done by Schnitzler et al. [15] shines light on the fact that when ALN is chemisorbed onto calcium deficient apatite (CDA), the increase in setting time was limited. They also observed ALN release to be constant, since it was a result of the interaction between ALN-CDA within the cement. The compressive strength increased as concentration of CDA increased for the two concentrations tested. CPC containing 0.1–0.3 wt.% ALN was formulated with properties suitable for minimally invasive delivery of this bisphosphonate locally.

3. Pamidronate

Cement powder made up of a mixture of 90% 𝛼-TCP and 5% HA with 5 wt% of DCPD (CaHPO₄ ⋅ 2H₂O, Merck) was created by Panzavolta et al. Cement paste was prepared by using a 0.3 mL/g ratio of liquid to powder. The bisphosphonate, pamidronate (PAM), as disodium salt was added in twice distilled water (liquid phase) at different concentrations of 0.4 and 1 mM. It resulted in initial setting times of 7 and 9 minutes for 0.4 mM PAM and 1mM respectively. The final setting times for those two concentrations were 25 and 27 minutes. In this study, the presence of bisphosphonates strongly impacted setting times, as initial and final setting times were found to increase with increasing concentration of bisphosphonate. It was also found that the conversion of 𝛼-TCP into CDHA is unaltered by bisphosphonate presence; all tested samples showed that the conversion is time dependent only. Compressive strength and the elastic modulus were reduced significantly for PAM0.4 and even more for PAM1.0 when compared to the control. This can be credited to the lesser affinity of PAM for hydroxyapatite, in addition to its higher steric hindrance, leading to a greater effect on the cements’ mechanical properties. The compressive strength for the PAM decreased significantly as the bisphosphonate concentration increased. In vivo studies have demonstrated that in both the PAM groups (0.4 and 1 mM) there is significant collagen (CICP) deposition, osteocalcin expression and alkaline phosphatase activity (ALP) compared to the control [16].

4. Zoledronate

Roussière et al. showed that a partial dissolution of 𝛽 – TCP caused a crystalline complex of calcium and sodium to precipitate. When washed with water repeatedly, this metastable phase was transformed into a calcium bisphosphonate complex. The bisphosphonate composite is poorly soluble in water. However, it was found that in a phosphate media, low doses of bisphosphonate directly proportional to the phosphate concentration are released. Based on these findings, it is feasible that the implantation of TCP+Bisphosphonate into bone tissue will cause a greater concentration of bisphosphonate to be released and more bone to be resorbed. This in turn creates a high release of phosphate into the ECM when the drug is required to decrease osteoclast activity [17].

An in vivo study was conducted on an osteoporotic rat model to study zoledronate (ZLN). CPC powders were crafted as indicated above with a few minor modifications. The powders were massed, packed in glass bottles, sealed and sterilized using an autoclave. ZLN was dissolved in distilled water, and 1 g of CPC powder was mixed either with 0.38 mL distilled water or ZLN solution forming a paste, 1.5 g of which was placed into a plastic plate. Blocks of ZLN/CPC/CaSO4 were prepared by adding CaSO4 into the powder phase, and afterwards, the blocks were placed in 10 mL phosphate buffered saline (PBS) at 37 °C with samples taken on day 1, 3 and 7. Four weeks post-ovariectom, rats under general anesthesia were implanted with the ZLN/CPC blocks. The ZLN release profiles revealed that the ZLN/CPC blocks had a parabolic release pattern, and after eight weeks 41.3 ± 4.8% of ZLN was released. Upon examination of the ZLN/CPC/CaSO4 blocks, the release pattern was found to be biphasic, and 54.9 ± 7.4% ZLN was released. Each of the rat’s limbs was harvested and subjected to micro-computer tomography (micro-CT) as well as bone ash analyses. There was no clinical toxicity observed in the treated rats. When comparing CPC rats, both the bone resorption marker (fragments of C-telopeptides of type I collagen) and bone formation marker (alkaline phosphatase and osteocalcin) levels were markedly lower in the treated rats. Also, markedly lower were the levels of osteopontin, which mediates the anchoring of osteoclasts to the bone mineral matrix. Examination of the scans from micro-CT and histological analysis of the distal femoral metaphysis discloses that the architecture of the cancellous bone was restored with a simultaneous decrease in the porosity of the bone in the treated rats. This study further shows that the mineral content in the bone ash also increased pointedly, and ZLN-infused CPC moderates bone turnover rate and restores bone architecture in CPC rats. These results give evidence to the theory that CPC may be an effective carrier of drugs that are used to treat osteoporosis, and that it may lessen the rates of post-dosing symptoms for intravenous delivery of ZLN [18]. A different in vivo study was conducted in which ZLN was released from preshaped calcium phosphate bone cement plugs loaded with the drug, and these plugs were implanted into the defective proximal tibia of rats. From this study and others, it can be seen that zoledronate containing implants improved contact between bone and implant, bone regeneration and bone density in cancellous rat bone compared to the control [19].

5. Etidronate

Etidronate is a bisphosphonate in which there is a central carbon with a hydroxyl group, configured to allow tridentate binding to calcium ions [20]. Nosoudi et al. investigated the addition of etidronate in HA (𝛽-TCP + CaCO3 + DCPA) cement using DI water by adding 1–50 μg etidronate disodium in the powder phase. The samples with different drug concentrations released about 20–40% of the drug, with the highest release value after 21 days for the 35 µg sample. Findings from this group showed that etidronate concentration resulted in higher initial and also final setting times to a point, followed by a drop-off, allowing for the optimization of the setting time by adjusting the etidronate concentration. The compressive strength of the W35 sample following 7 days was the greatest, and the data showed significant decrease in the compressive strength when the content of etidronate exceeded 35 μg. The study concluded that increasing etidronate would consistently boost initial setting time until it reached a maximum. This was attributed to the Ca²⁺ chelation induced by the bisphosphonate, which delayed the initial setting [21].

6. Future directions

As of yet, there are limited ways to obtain the desired setting time for CPCs, because the setting time of brushite CPCs is generally too short and that of apatite CPCs is generally too long [22,23]. However, bisphosphonates may be a solution, as all that have been tested have slowed setting time in CPCs. Nosoudi et al. found that etidronate slowed the setting of CPC in low concentrations, while also speeding it up in high concentrations [21], and Schnitzler et al. found that a larger amount of ALN effectively slowed the setting speed when it was loaded in the cement paste. Bisphosphonates were mostly found to have retarding properties in regards to setting time, but this clearly varied based on the conditions and were diminished in the presence of CDA [24]. The findings are summarized in Table  1. It appears from the data that bisphosphonate has dual behavior, because in addition to the well documented retardant affect, it accelerates the setting when a very low amount is added. We know there is a clear dependency between release rate and cement porosity which can be easily controlled by modifying the liquid-to-solid (L/S) ratio which is valid for composites of calcium phosphate-bisphosphonate as well. A lower L/S ratio leads to lesser porosity and longer release [25]. Also, as we expect, higher L/S increases the setting time [26].

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Table 1 (Continued).

As far as the mechanical properties are concerned, the addition of residronate into calcium silicate cement negatively affects its mechanical properties, as seen in previous studies. This phenomenon likely can be attributed to the HA formation inhibitory effect [27]. A similar result was observed in etidronate cement composite before soaking it in the ringer solution [21]. However, a study conducted on PAM/CPCs composites showed that the stiffness and Young’s modulus of the bisphosphonate-enriched bone cement in the compressive test were decreased by less than 5% compared to the control. According to these findings, the most important mechanical properties of BP-enriched cement were preserved [28]. Shen et al. showed that there is an advantage to adding ALN, which was to increase mechanical strength. When the content of ALN was less than 1.0 wt%, the pores were fine and small so the structure remained compact and the compressive strength increased. Increasing ALN content more than 1.0 wt% causes bigger pores to form; thus, the comprehensive strength decreases [14].

Hydroxyapatite formation and dissolution are two key phenomena that are affected by bisphosphonate addition. Bisphosphonates, polyphosphates or pyrophosphoric acid are all responsible for inhibiting HA dissolution [29]. The explanation for this comes from the striking affinity of these three compounds to the solid-phase calcium phosphate surface, as they can bidentate or tridentate bind to the calcium by chemisorption. In bidentate binding, as seen in clodronate, an oxygen atom from the two phosphonate groups binds to calcium in the hydroxyapatite. However, trindentate binding is the most common among bisphosphonates. Tridentates bind at an additional third position, such as an oxygen belonging to a hydroxyl group on the central carbon, imparting their name. Tridentate binding exhibits better binding strength, explaining the infrequent binding habits of clodronate. Sometimes, the hydroxyl group is replaced by a nitrogen atom, as in incadronate. Bisphosphonates binding and inhibiting crystallization is reasoned by a theory which states that the inhibition of mineralization in vivo is because of a physicochemical mechanism. So far it is undetermined whether bisphosphonates get incorporated into the crystal lattice of hydroxyapatite, but it is confirmed that hydroxyapatite is assimilated into the bone since it is crystalline, and bisphosphonate on the surface gets trapped by fresh crystals forming on top of them. Over all, reduced bone resorption and bone formation, evaluated in animals and humans by calcium kinetics, biochemical markers, and morphological cues validates that the action of active bisphosphonates is consistent for all types [18,30].

Low ALN sodium concentration has been shown to make the environment more acidic and delay the transformation into HA. Higher concentrations of ALN sodium seemingly promote HA production. The delaying or accelerating effect of various ALN sodium concentrations affects nanocrystal growth which determines the final phase [31]. A CPC-etidronate composite was studied to substantiate these results. From the study results, in day 1, samples with higher etidronate concentration had less apatite in comparison to samples with less etidronate, but this changed after 14 days, where the apatite content in samples with higher etidronate was higher than samples with less etidronate. Etidronate occupies the active sites initially, so it prevents apatite formation in high content samples, but after time, facilitates apatite formation [21].

At this point in time, most in vivo studies focus on ZLN or ALN-CPC composites. There is a distinct lack of studies on other CPC-BP composites, creating a large area for potential developments and applications of these materials. Potential to study other bisphosphonate composites, such as neridronate, olpadronate, ibandronate, incadronate and others in vitro also offer huge possibilities [32].