Effects of Adding Resorbable Phosphate Glass Fibres and PLA to Calcium Phosphate Bone Cements

Abstract

Background

Calcium phosphate cements (CPCs), due to their biocompatibility and degradation properties, are being widely investigated as a replacement to more commonly used polymethylmethacrylate (PMMA) for vertebroplasty. CPCs have shown the potential to be replaced by host bone tissue during the healing/remodelling process. However, brittleness and comparatively low strength restrict the use of CPC in load-bearing applications. Although porous CPC can integrate with bone over time, slow degradation profiles and poor interconnectivity between pores restricts osseointegration to the top layer of CPC only.

Methods

Polylactic acid (PLA) and phosphate glass fibres (PGFs) were incorporated in a CPC matrix to overcome the problem of inherent brittleness and limited osseointegration.

Results

Incorporation of PLA and PGFs within CPC was successful in achieving a much less brittle CPC matrix without affecting the mechanical properties of CPC. The area under the stress-strain curve showed that the total energy to failure of the CPC hybrid was significantly greater than that of the CPC control.

Conclusions

The methodology adopted here to add PLA within the CPC matrix may also allow for incorporation of PLA cross-linked biochemicals. Micrographic studies revealed that it was possible to confer control over pore size, shape and interconnectivity without negatively affecting the mechanical properties of the cement. This tailorable porosity could potentially lead to better osseointegration within CPC.

Keywords

Calcium phosphate cements Phosphate glass fibres Polylactic acid PLA

Introduction

Vertebroplasty is a quick, minimally invasive surgical procedure which stabilises vertebral compression spinal fractures by injecting a self-setting paste of bone cement into the fractured vertebral body (1-3). The current standard bone cement material used is polymethylmethacrylate (PMMA), which has proven to be effective in vertebroplasty and kyphoplasty procedures, as shown by clinical results (2, 3). Other benefits of PMMA include ease of handling, high mechanical strength and low cost. However, PMMA does not allow bone ingrowth. PMMA can cause tissue necrosis due to high polymerisation temperature (up to 113°C) (4). Potential toxicity from the monomer, if it leaches out, has also been reported (4, 5). PMMA can also increase the risk of fracture to adjacent vertebral bodies due to the mismatch of stiffness properties between PMMA-reinforced and adjacent vertebral bodies (6-8).

Calcium phosphate cements (CPCs) are widely being investigated as a replacement for PMMA due to their biocompatibility and degradation properties. CPC can also be replaced gradually by host bone tissue during the healing/remodelling process (7, 9, 10). Nonetheless, the benefits of CPC are overshadowed by its brittleness and relatively low strength, which restricts the use of CPC especially in higher load bearing applications (11). CPC generally has porosity values between 30%-60% of volume, depending on the composition, which can have a detrimental effect on the mechanical properties of the cement (as a rule of thumb, tensile strength can increase twofold with a 10 volume% decrease in porosity) and also on the degradation rate (11).

To control cement porosity and incorporation of drugs or other bioactive agents within the cement matrix, degradable polymer particles or microspheres have been incorporated (9, 12). For example, poly(lactic-co-glycolic acid) (PLGA) microspheres were successfully incorporated with a CPC system, which resulted in enhanced apatitic CPC degradation; however, no mechanical properties were reported (12). Similarly, chitosan particles (13), gelatin microspheres (14), maleic acid or polyacrylic acid (15) and polyamide (16) have also been incorporated. Mechanical properties of CPCs are not narrowly distributed around a mean value, but cover a very large range. In addition to large deviations, different formulations of CPC exhibit compressive strength values in the range of 10 to 100 MPa (17). However, despite the difference in formulations of CPC and the polymer incorporated, most of the studies reported a significant improvement in either flexural or compressive strengths. For example, an α-tricalcium phosphate (α-TCP)-based CPC was mixed with polyamide, 6×12 mm cylindrical samples were tested for compressive strength and an improvement of 3.5 MPa (from 9.5 MPa to 13 MPa) was reported (16). Another study reported improvement from 17.4 MPa (for tetra calcium phosphate [TTCP]– and dicalcium phosphate [DCP]–based CPC) to ~90 MPa with polyacrylic acid incorporation (18). The improvements were attributed to noncrystalline hydroxyapatite (HA) formation, greater viscosity and lower porosity.

Reinforcement of CPC with fibres to improve the initial strength and fracture resistance of the cement has also been investigated. The variety of fibres used to reinforce CPCs have included polylactide, polyamide, polyester (poly-ε-caprolactone and polyglycolide), Kevlar, carbon and E-glass fibre (19). In a specific study, Vicryl™ (polyglactin 910; Ethicon, Somerville, NJ, USA) and Vicryl Rapide™ (polyglactin 910; Ethicon) fibres were used as reinforcement. The results showed decreased yield strength due to increased porosity, and consequently reduced dynamic loading of CPC (20). However, the strength reduction can be compensated for by the excellent bone biocompatibility of CPC, which allows it to be replaced by the host bone (6). Research has shown that fibre type, length and volume fraction were crucial factors in determining mechanical properties of reinforced CPC (21). According to dos Santos et al (16), fibre reinforcement of CPC with polyamide fibres of up to 1.6% showed little effect on improving the compressive strength of the cement. Another study by Xu and Simon (22) incorporated adsorbable mesh sheets of interconnected polyglactin fibres into CPC to increase the strength by threefold and the work of fracture (WOF) by 150 times. Buchanan et al (23) investigated the use of polypropylene fibres, and their results showed that a fibre volume fraction of up to 10% had a significant effect on cement mechanical properties, with specimens showing values of up to 7 MPa and 200 MPa for compressive strength and modulus, respectively.

Synergistic approaches have also been investigated where the degradable fibre and polymer particles have been incorporated together in a CPC (24, 25). For example, suture fibres and chitosan were incorporated with TTCP/DCPA based cement. Flexural strength (3-point bend test) was reported to increase from 2.7 MPa to 11 MPa with addition of chitosan, to 18 MPa with suture and to 40 MPa with chitosan and suture together synergistically (22). In another study, flexural strength and flexural fatigue properties were found to improve with the incorporation of chitosan particles and polygalctin fibres (25). Flexural strength (3-point bend) improved from ~10 MPa to ~25 MPa, while hybrid CPC-chitosan fibre specimens survived more than 2×10⁶ cycles (4-point flexural fatigue) without fracture, and failed at higher stresses than CPC control (25).

In this study, (α-TCP)-based CPC was prepared and reinforced with phosphate glass fibres (PGFs), polylactic acid (PLA) or synergistically with PGF and PLA. Phosphate glass formulations can easily be altered to adjust their degradation rates from hours to days to years (26, 27). For the purpose of this study a PGF was selected for its strength, biocompatibility and control over degradation rate (26, 27), whilst PLA was selected to exploit its effect on CPC fracture. Cylindrical specimens (diameter × length=6×12 mm) were prepared and compression tested to failure according to ISO 5833.

Materials and Methods

CPC Specimen Preparation

CaCO₃, Ca(H₂PO₄)₂.H₂O and (NH₄)₂HPO₄ were bought from Sigma Aldrich UK and α-TCP was acquired from Plasma Biotal Ltd. All chemicals were analysed using X-ray diffraction (XRD) and used without further purification.

CPC in its solid phase was prepared by mixing 85 wt% α-TCP, 12 wt% CaCO and 3 wt% Ca(HPO)₂.H₂O. A homogenised solid phase mixture was then supplemented by 3.5 M (NH₄)₂HPO₄ liquid phase in a ratio of 1.2 g:1 mL. The resultant paste was cured at 37°C for 24 hours and ground to powder; this was referred to as CPC preset. CPC-preset powder was admixed in the same liquid phase at a ratio of 1.5 g:1 mL. The ensuing paste was inserted into a cylindrical polytetrafluoroethylene (PTFE) mould with inner dimensions of 6×20 mm (diameter × depth) and left to cure at 37°C for 48 hours. These samples were labeled as CPC control in this study.

For PLA additions to CPC, a predetermined (1-5 wt% of CPC preset) amount of PLA (3251D Naturework Plc.) was dissolved in chloroform, which was then mixed with the CPC-preset in its solid phase. After chloroform had evaporated, dried PLA/solid phase was ground by pestle and mortar, mixed with the above-mentioned liquid phase mixture and cured at 37°C in the PTFE mould for 48 hours and was labeled as CPC-PLA.

PGFs of ~20-μm diameter were prepared using an inhouse fibre production facility, and chopped to 2.5 mm length (see (27) for further details on fibre production). A predetermined amount (1-5 wt%) of chopped PGFs was mixed with the CPC-preset in its solid phase. The same liquid phase mixture was added to the PGF/solid phase mixture and cured at 37°C and was referred to as CPC-PGF.

PLA/solid phase was prepared, and a known amount (1-5 wt%) of PGFs was added to prepare synergistic samples containing CPC preset, PLA and PGF. These hybrid samples were also mixed with the same liquid phase and cured at 37°C for 48 hours. These specimens were labeled as CPC hybrid.

Compression Test

The samples were cut to size (diameter × length = 6×12 mm) in accordance with ISO 5833:2002 for compressive tests. Prior to testing, the end surfaces of all samples were polished using sand paper (P240 with average particle size of 58 μm). The compressive strength was measured using a Hounsfield Series S testing machine fitted with a 1-kN load cell configured to compress at a rate of 1 mm/min.

SEM Analysis

Cross-sections of the CPC samples, exposed after compressive fracture, were sputter coated with platinum and analysed using a Jeol XL 30 scanning electron microscope (SEM; Philips, UK) at an accelerating voltage of 10 kV and approximate working distance of 10 mm.

Results

Figure 1 (a-c) presents compressive strengths for CPC control, CPC-PLA, CPC-PGF and CPC hybrid, with increasing amount of PLA or PGF or their combination.

Fig. 1

Compressive strengths and moduli for calcium phosphate cement (CPC) with increasing a) polylactic acid (PLA), b) phosphate glass fibres (PGFs) or c) combination of both. Weight percentages of fillers (PLA or PGF) are included. Error bars represent standard error of the mean. Number of samples per test = 6.

An upward trend in compressive strength of CPC was found with addition of PLA, which improved by ~1 MPa with 5% PLA doping, compared with control. Conversely, a drop in strength was observed with addition of PGFs. A lower value of ~5.5 MPa was observed with higher amounts of PGFs. Statistical analysis revealed no significant difference (P>0.05) between any of the samples tested. Moduli in the range of approximately 400 MPa to 600 MPa were recorded for different CPC samples. A trend of reduction in modulus with PLA and increment with PGF was also observed.

Figure 2 shows comparative load–displacement curves obtained for 4 types of CPC samples. A highly brittle failure mode can be observed for CPC control and CPC-PGF in contrast to CPC-PLA and CPC hybrid, where a less brittle failure mode is observed. The curves clearly demonstrate that PLA-doped samples absorbed more energy than CPC control and CPC-PGF.

Fig. 2

Representative load versus displacement curves for 4 types of calcium phosphate cement (CPC) samples, showing highly brittle failure mode for CPC control and CPC–phosphate glass fibre (PGF) in contrast to CPC–polylactic acid (PLA) and CPC hybrid, where a less brittle failure mode is observed.

The area under the stress-strain curve, up to the point of failure, represents the total mechanical energy per unit volume absorbed by the material. The stress-strain curve was recorded until the collapse of the specimen. The area under the curve (Fig. 3) was calculated for CPC control and CPC hybrid using Origin software (OriginPro 7). For these particular samples, the energy absorbed was found to be 73 mJ for CPC control and 129 mJ for CPC hybrid. Comparison of more samples from CPC control and CPC hybrid/CPC-PLA revealed ~40% more absorbed energy for PLA-doped samples with similar compressive strength. Images (Fig. 4) of the samples taken during compression testing show the failure modes that occurred for CPC control, CPC-PGF, CPC-PLA and CPC hybrid.

Fig. 3

Comparison of energy absorbed (area under the curve) for load displacement plots for calcium phosphate cement (CPC) control (73 mJ) and CPC hybrid (129 mJ).

Fig. 4

Images of the samples investigated during compression testing to failure: a) calcium phosphate cement (CPC) control, b) CPC–phosphate glass fibre (PGF), c) CPC–polylactic acid (PLA) and d) CPC hybrid.

Figure 5 (a-d) shows representative SEM micrographs of the samples investigated. Large voids and loosely packed CPC matrix were observed for the CPC control and CPC-PGF. For CPC-PGF, the voids appeared to be concentrated around the fibres. Incorporation of PLA with CPC revealed a much denser CPC matrix, as was the case for the CPC hybrid. Partially degraded fibres (probably due to the liquid phase of the CPCs) can also be observed in CPC-PGF and CPC hybrid, which would eventually degrade in vivo to leave behind channels through the CPC matrix.

Fig. 5

SEM micrographs of a) calcium phosphate cement (CPC) control, b) CPC–phosphate glass fibre (PGF), c) CPC–polylactic acid (PLA) and d) CPC hybrid. Micrograph scale bar = 100 μm.

Discussion

Calcium phosphate cements (CPCs) can be injected directly into fractures and bone defects, where they adapt to the bone cavity regardless of irregularities in bone shapes. CPCs can provide a number of advantages such as the development of osteoconductive pathways, adequate compressive strengths and chemical bonds to the host bone, and they have both chemical composition and X-ray diffraction patterns similar to those of bone (28). They can also be regarded as being osteotransductive – i.e., replaced by new bone tissue over time (2, 29). A number of excellent reviews on the compositions, mechanical and biological properties of CPCs have been published (19, 30, 31).

The CPC formulation emulated in this study was based on the Norian SRS^®–based calcium orthophosphate cement formulation approved by the US Food and Drug Administration (7, 31, 32), which set according to 2 chemical reactions (Eqs. 1 and 2): precipitation of dicalcium phosphate dihydrate (Eq. 1; cement setting within seconds), followed by precipitation of either calcium-deficient hydroxyapatite or carbonate apatite (Eq. 2; cement hardening takes hours):

α - {Ca}_{3} {({PO}_{4})}_{2} + Ca {(H_{2} {PO}_{4})}_{2} • H_{2} O + {7H}_{2} O \to {4CaHPO}_{4} • 2 H_{2} O

[1]

5.2 {CaHPO}_{4} \cdot 2 H_{2} O + 3.6 {CaCO}_{3} \to {Ca}_{8.8} ({HPO}_{4}) 0.7 ({PO}_{4}) 4.5 ({CO}_{3}) 0.7 {(OH)}_{1.3} + 2.9 {CO}_{2} + 12 H_{2} O

[2]

Norian SRS^® forms a nonstoichiometric carbonate apatite or dahllite as the end product due to presence of carbonates (33). Owing to formation of calcium-deficient hydroxyapatite and carbonate apatite having low crystallinity they appear to be similar to biological apatite of bone. The setting time of a CPC can be adjusted by using additives such as phosphoric acid, Ca(H₂PO₄)₂ • H₂O or other soluble orthophosphates such as Na₂HPO₄ or NaH₂PO₄. A list of compounds, employed as accelerators, retarders, additives or reactants in calcium orthophosphate cement formulations, can be found in these references (34, 35). These additives promote dissolution of the solids by lowering the solution pH. In such cases, a setting time in the range of 10-15 minutes can be obtained (34, 35). After unsuccessful trials with Norian SRS^®-specific NaH₂PO₄, (NH₄)₂HPO₄, which is also reported as a setting time accelerator (35), was used.

CPCs are well known to be brittle and hence of limited use for load-bearing applications (19, 23). Slow degradation is another problem associated with CPC. For example, only a 30% decrease of the implanted amount of Norian SRS^® was reported after 24 months in a rabbit femur (29). Therefore resorbable PLA and PGF were incorporated to improve the ductility and gain control over resorption profiles of CPC.

Different CPC samples produced here demonstrated a compressive strength of approximately 8-10 MPa (Fig. 1), which was comparable to the similar composition (61% α-TCP; 26% CaHPO₄ and 10% CaCO₃) incorporated with hydroxyapatite and/or polypropylene fibres (23). A maximum of ~10 MPa (control) compressive strength was reported, which decreased to ~7 MPa with increasing polypropylene fibre fraction to 10% (23). Another study (16) of α-TCP-based cement reinforced with short polyamide fibres reported a compressive strength of ~10 MPa for control which improved slightly (0.2% to 0.8%) with incorporation of these nondegradable polymeric fibres. Maximum compressive strengths of 50 MPa for the Norian SRS^®-injectable system (36) and ~30 MPa for Norian SRS^® fast setting putty (37) have been quoted. The difference observed in the current study could be due to high porosity caused by difference in setting accelerator used and different powder to liquid ratio. Compression strength is reported to be reciprocally proportional to porosity (38). Release of ammonia gas from (NH)₂HPO₄ used in the current study could have introduced greater porosity within CPC and as a result decreased the strength of CPC.

A synergistic effect was achieved by reinforcing CPC with both PLA and short PGFs, where PLA improved the ductility and PGF could potentially provide channels for osseointegration as they were resorbed. In addition, the resorption of PGFs would release calcium and phosphate ions which could assist in the bone repair process. CPCs can be degraded by active or passive resorption. Active resorption is a cellular process mediated by osteoclasts, whilst passive resorption is based on the chemical solubility of the CPC material. The introduction of controllable porosity and pore interconnectivity into CPC could favour cell penetration as well as control over degradation rate (12).

Fracture behaviour of different CPC samples (Figs. 3 and 4) revealed that incorporation of PLA improved the ductility of CPC. It was reported in the literature that incorporating short polymeric fibres in the solid phase improved the ductility of CPC (23, 25). However, incorporating 15% chitosan (by weight) in its liquid phase did not improve the ductility (25). The results presented in the current study demonstrated the effectiveness of the approach adopted to incorporate PLA in the solid phase of CPC. The energy absorbed (Fig. 3) by CPC-PLA and CPC hybrid was almost double that for the CPC control. Moreover, the fracture behaviour (Fig. 4) was found to be less brittle with incorporation of PLA.

It has been reported that long cylindrical pores are advantageous for osteoconduction and active bone ingrowth compared with random and spherical pores (39). Small interconnections between pores do not permit cell migration or tissue growth, thus limiting tissue ingrowth to a shallow surface layer of the implant (39, 40). CPC can be resorbed and replaced by new bone, hence its macropore and interconnection sizes are expected to grow over time in vivo. The addition of resorbable fibres could increase cement porosity as the fibres dissolve over time creating macropores and controllably sized cylindrical channels, thus allowing for potential bone ingrowth to occur. Channels created by PGF can be observed in micrographs (Fig. 5).

If filler is present in the CPC, it may possibly prevent crack propagation. However, adding fillers reduces the porosity and slows down resorption, which results in slower bone substitution (38). Incorporation of PLA was found to increase the density of CPC (Fig. 5). Injectability and flow rate of CPC also worsen with the addition of fillers (19, 30, 41). In the current study, injectability of CPC was found to worsen with incorporation of higher amounts (10%) of PGF alone. However, CPC-5%PLA and selected CPC hybrid (5%PLA + 5%PGFs) were injectable, with a potential control over porosity and interconnectivity between pores via the degradation rate of the PGFs. The degradation rate and fibre diameter can easily be controlled by modifying glass composition and fibre drawing rates, respectively (26, 27).

A potential drug delivery device must exhibit the ability to incorporate a drug, retain it in a target site and deliver it in a controlled manner to the surrounding tissues. Injectability, low setting temperature and near neutral pH allow the incorporation of different drugs within CPC-PLA making them an attractive candidate as drug carriers (42). Incorporation of PLA within CPC could provide the added benefit of additional control over drug release via cross-linking or encapsulating drugs or other bioactive agents (i.e., growth factors or proteins) within the degradable polymer.

In summary, this pilot study developed a less brittle (ductile), injectable CPC with potentially controllable porosity (via PG fibre resorption). No statistically significant difference was observed via the addition of fillers (PLA or PGF) on the mechanical properties of CPC. However, further investigations need to be conducted with varying molecular weights and amounts of polyesters (PLA, PGA and poly-ε-caprolactone) along with PGF volume fractions. In addition, cyclic fatigue testing to investigate mechanical performance over many cycles will also be required.

Conclusions

Incorporation of PLA and PGFs within CPC were successful in achieving a much less brittle (i.e., ductile) CPC matrix. The data obtained indicated that with PLA incorporation, although the CPC matrix was much denser and less brittle, higher amounts of PGFs reduced the injectability. The area under the stress-strain curve showed that the total energy absorbed by CPC hybrid was significantly greater than that by CPC control.

SEM studies revealed that it was possible to confer control over pore size, shape and interconnectivity via the fibre degradation profiles without negatively affecting the mechanical properties of CPC. This tailorable porosity could potentially lead to better osseointegration within CPC.

Footnotes

Financial support: This work was supported by a Nottingham University Hospitals NHS Trust Innovation grant and the Biocomposites Research Group at the University of Nottingham.

Conflict of interest: None.

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