Application of ES loaded PMMA Bone Cement
It has been over 90 years since the discovery of heparin as an effective anticoagulant. The mechanism of anticoagulant is to activate antithrombin III (ATIII) by the allosteric effect through the specific combination of polysaccharide sequence and ATIII [12-14]. The activated ATIII can inhibit a variety of coagulation factors including Xa, IIa, IXa, XIa, XIIa etc., to exert the anticoagulant activity. Researchers have been working on the improvement of heparin to overcome some of the inherent shortcomings, such as short biological half-life, risk of bleeding, and the need of blood coagulation monitoring. Enoxaparin is a low molecular weight heparin produced using the ordinary heparin as the raw material and obtained by β- elimination degradation. Compared with ordinary heparin, enoxaparin has a strong antithrombotic effect, weak anticoagulant effect, low incidence of side effects such as bleeding, good absorption of subcutaneous injection, long biological half-life and predictable anticoagulant effect. At present enoxaparin has replaced the ordinary heparin in clinical practice [15-18]. Enoxaparin sodium has an average molecular weight of 3800-5000, anti-FXa activity of 90 IU/mg-125 IU/mg, anti-FIIa activity of 20 IU/mg-35 IU/mg, anti-FXa/anti-FIIa ratio of 3.3-5.3, and an excellent water solubility .
Principles of PMMA Bone Cement as Drug Carriers
PMMA bone cement has been widely used in clinical practice, such as total hip replacement, half hip replacement, total knee replacement or single patella replacement, vertebroplasty, bone tumors, pathological fractures, spacers with PMMA bead chain during infection, or placeholders for soft tissue defect in open fracture, etc. [20-24]. As the drug carrier, the most mature and largest scale commercial application of PMMA bone cement is antibiotic loaded PMMA bone cement [25-28]. From the experience of antibiotic loaded bone cement, certain physical and chemical properties are required to facilitate the effective release of drug from bone cement and act on the body. These properties include high water solubility, resistance to radiation disinfection or epoxy ethane sterilization, stability when stored with bone cement powder before use, inactivity during bone cement polymerization, high temperature resistance, little or no effect on mechanical strength of bone cement, and good release from solid bone cement [29-34]. Stevens et al.  reported that the characteristics of drug release varied a lot when using different brands of bone cement. The best release characteristics were tested from Palacos®R bone cement. Another bone cement Simplex®, which has been widely used in clinical practice, exhibited poor release characteristics. The bone cement with good release characteristics may have more pores and looser structure among the polymer beads after polymerization, thus more amount of drug can be loaded and the contact surface with solution is larger. Based on the above reasons, the Palacos®R bone cement was selected as the ES carrier in this study.
The surface of no ES loaded bone cement was observed under the SEM. Most of the beads observed were large and uneven copolymers, which are the polymethyl methacrylate copolymers manufactured by industrial grinding. These beads were bound together by PMMA during the polymerization reaction of methyl methacrylate (MMA). There were pores of various sizes between the polymer beads. The entire PMMA bone cement formed a sponge-like three-dimensional structure which serves as the structural basis for drug loading.
Syrup-like substances between the copolymer beads were observed on ES loaded PMMA bone cement. With increased ES loading amount, the syrup-like substances also increased and wrapped on the surface of copolymers beads like sugar coating. Comparison between the SEM photos before and after drug release revealed that the syrup-like substances were reduced significantly. We inferred that these substances were ES, a highly sulfated glycosaminoglycan. After drug release the pore space became large. The super large pores with diameters > 500um were observed in multiple SEM photos. By providing channels for capillary growth and an environment and framework suitable for cell growth, these pores enhanced the biocompatibility of drug-loaded bone cement and mechanical strength in connection with the surrounding tissues [36,37].
Mixing Process of ES-PMMA bone cement
There are two mixing methods in the drug-bone cement release system. One is to thoroughly and evenly mix the drug lyophilized powder or fine particles with bone cement powder component. The liquid component is poured into the mixture before use and evenly mixed vacuumed or manually. The other is to thoroughly and evenly mix the drug lyophilized powder or fine particles with bone cement powder component. The drug is added in the dough phase so that the drug is wrapped within the bone cement dough [38, 39]. The first method is used in various antibiotic loaded bone cement on the market, such as Palacos®R+G, CMW®1G etc. These mature products are all prepared using this way in order to make the drug component evenly distributed inside the bone cement and make the product more convenient to use [40, 41]. The second method is more used in the operating room and prepared manually. The advantages include various amount of drug loading and specific individual treatment. However, the disadvantages are obvious. The uneven mixing has a great impact on bone cement mechanical strength and uneven drug distribution makes it hard to predict the release characteristics [42-44]. In order to minimize the influence of human factors on the release characteristics of bone cement, the first mixing method was used. The ES lyophilized powder was fully ground into fine particles, mixed with bone cement powder thoroughly until no drug particles are visible to the naked eye and then mixed with the liquid components of bone cement to make the test mold for the experiment.
ES-PMMA Bone Cement Release Characteristics
The release mechanism of the drug-bone cement release system follows the principle of dispersion. Dry bone cement absorbs water, and water-soluble drugs are released with the random and irregular thermal motion of water molecules . The factors which affect the drug release rate from the bone cement include: 1) absorption of water, surface area and porosity of bone cement, 2) nature and content of drug and 3) mixing method [46-48]. Faster water absorption, larger surface area and higher porosity lead to a higher drug release rate and release amount. Regarding the effect of drug nature and content on drug loaded bone cement release system, Kuehn et al.  reported that the particle size of drug affects the release amount under the comparable conditions of PMMA bone matrix, operating separation, mixing techniques, and drug content. The release amount of coarse drug particles is higher than that of fine drug articles, which is higher than that of very fine drug articles. The drug release amount of manual mixing is greater than that of vacuum mixing when the other conditions are comparable. This is because vacuum mixing may decrease the porosity of PMMA bone cement, and drug is released from the pores of bone cement matrix. With less pores, drug release is decreased [50, 51]. Despite careful grinding of the ES lyophilized powder in the mortar in the experiment, the drug particles were very coarse while the bone cement powder particles were very fine. There was visible difference between the two and it was not obvious until the two particles were completed mixed. The liquid bone cement was mixed carefully with the powder mixture for 30 seconds manually following the user instruction of Palacos®R bone cement to obtain the ES loaded bone cement for experimental use. The test mold prepared in this way exerted higher release amount and a faster release rate than the product manufactured in ideal conditions (prechilled super fine particles of ES and bone cement powder mixed under vacuum).
The mold used for the preparation of the test mold was made according to ISO5833:2002 “Surgical Implant – Acrylic Resin Bone Cement”. As shown in Figure 7, computer aided design was used for mold drawing and mold was made by 3D printing using photosensitive resin. The test mold was a cylinder with a length of 12±0.1mm and a diameter of 6±0.1mm, with regular shape and smooth surface, as shown in figure 8. This test mold is quite different with the shape of bone cement implanted in human body in clinical practice. In the actual clinical application, the solidified bone cement closely adheres to the inner wall of the medullary cavity or the bone surface, with an irregular shape and rough surface. These features increase the surface area of bone cement [52, 53]. Riva et al.  found that most of the drug in the drug-bone cement release system cannot be released. The drug is released from a thin layer on the bone cement surface. Release amount is proportional to the surface area of the bone cement. Thus, the larger surface area per unit volume of bone cement, the more the drug is release. Therefore, we believe that the release amount from the actual drug loaded bone cement system implanted in the human body is greater than that of the in vitro release system.
The Release Effect of ES-PMMA Bone Cement
The absorbance value of extract of ES bone cement can be obtained by the chromogenic substrate method. The value was converted into a logarithm. According to the quantitative response parallel line method documented in Chinese Pharmacopoeia 2015, Appendix 1431, the 4.4 method was used to calculate the potency and experimental error and to depict the release curve. The curve showed that the ES was released at a high concentration and reached the peak on the first day. The release then rapidly decreased to a low range and became stable. Consistent with the results reported by Anguita-Alonso et al. , our results showed that the drug-bone cement release system generally has a burst effect and the sustained release may last for a long time. The anti-FXa activity for the therapeutic effect of ES to prevent thrombosis is 0.20-0.50 AxaIU/ml and may reach 1.0 AxaIU/ml under the therapeutic amount. In the experiment with 4000 AxaIU ES added to 40g PMMA bone cement, the release amount reached the therapeutic dose (about 0.40 AxaIU/ml) within 24h. With 8000 AxaIU ES loaded to 40g PMMA bone cement, the release amount reached the therapeutic dose (about 0.40 AxaIU/ml) within 24h and drug concentration maintained at the range between the preventive dose and therapeutic dose. With addition of 8000 AxaIU or more of ES, the drug centration released in 24h exceeded the maximum therapeutic dose of anticoagulation, which may cause a bleeding risk. Therefore, it is recommended to choose the ES amount within a safe drug concentration.