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Structural control and hemostatic properties of porous microspheres fabricated by hydroxyapatite-graft-poly(D,L-lactide) nanocomposites
Composites Science and Technology 134 (2016) 234e241
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Structural control and hemostatic properties of porous microspheres fabricated by hydroxyapatite-graft-poly(D,L-lactide) nanocomposites Leyuan Song a, Lei Sun b, Ni Jiang a, *, Zhihua Gan a, ** a State Key Laboratory of Organic-Inorganic Composites, Beijing Laboratory of Biomedical Materials, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China b Beijing Institute of Traumatology and Orthopaedics, Jishuitan Hospital, Beijing 100035, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2016 Received in revised form 31 August 2016 Accepted 1 September 2016 Available online 1 September 2016
Nanocomposite microspheres with tunable porous structures were studied in this work as hemostatic agents. Hydroxyapatite nanoparticles-graft-poly(D,L-lactide) (nHA-g-PDLLA) nanocomposites were synthesized first and then fabricated into porous microspheres by a modified double emulsion solvent evaporation technique. Alkaline treatment were carried out on nanocomposite microspheres for the exposure of HA nanoparticles on the surface of porous microspheres. The porous structure and nanocomposition endue the nHA-g-PDLLA microspheres with huge specific surface area and excellent interfaces for blood clotting. This work reveals the great potentials of nHA-g-PDLLA nanocomposite microspheres as hemostatic agents. © 2016 Published by Elsevier Ltd.
Keywords: Polymer-matrix composites (PMCs) Interface Surface treatments
1. Introduction Uncontrolled massive hemorrhaging has always been the leading cause of deaths after injury whether in war or daily life [1e3]. In fact, natural response to injury of human body can deal with light wound with three stages, i.e., the formation of a platelet plug, an enzymatic cascade resulting in the formation of fibrin, and the dissolution of clot and healing of wound site [4,5]. Therefore, tremendous hemostatic agents have been developed to intervene in the coagulation cascade for arresting bleeding and stabilizing wound. Current hemostatic agents approved clinically act through passive, active or a combination of mechanisms [6]. For example, oxidized/oxidized regenerated cellulose, gelatin sponges, collagen pads and sponges play the passive hemostatic roles through surface acting, while thrombin, fibrin sealants and hemostatic patches are active ones that partake in the coagulation cascade by providing biologically active components. Great efforts have been made to develop novel composite hemostatic agents that combined passive and active components. Polyphosphate refined the chitosan dressings, calcium-modified microporous starch, and graphene contained chitosan-poly(vinyl alcohol) nanofibers have been
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (N. Jiang), [email protected] (Z. Gan). http://dx.doi.org/10.1016/j.compscitech.2016.09.001 0266-3538/© 2016 Published by Elsevier Ltd.
reported to be potent wound dressings or beneficial for wound healing [7e9]. Hydroxyapatite (HA, Ca10(PO4)6(OH)2), which is the main inorganic component of natural bone, has been widely studied in bone tissue engineering and industrial catalysts [10,11]. It has been reported that HA nanoparticles possess an extraordinary adsorption of proteins due to the surface charge and particle texture [12]. The easy protein-binding surface of biomaterials is favorable for the platelets adhesion and the blood clotting formation due to the adsorbed proteins in blood coagulation cascade [13]. Moreover, HA 2þ exhibits both acid (PO3 ion) active sites based 4 ion) and base (Ca 2þ on its crystal structure [14,15]. The Ca is an important factor in blood coagulation cascade, named the fourth clotting factor which is also the only inorganic factor [16,17]. It has been reported that the addition of Ca2þ ions not only decreases the induction period of the fibrinogen-fibrin system in the coagulation cascade, but also greatly promotes the fibrin monomer polymerization [18]. Therefore, the calcium-modified microporous starch prepared by oxidization and self-assembly with Ca2þ [8] has been reported to own potent hemostatic efficiency for hemorrhage control because of the acceleration of Ca2þ for blood clotting. Microspheres fabricated by synthetic biodegradable polymers such as polylactide (PLA), its copolymer with polyglycolide (PLGA), and poly(ε-caprolactone) (PCL) are widely studied for tissue engineering and drug delivery. The microspheres are usually prepared by emulsion solvent evaporation techniques [19]. However, because
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of the lack of bioactive sites and the intense hydrophobicity, great efforts have been made in fabricating composite microspheres to meet the versatile requirements in application [20]. In consideration of the bioactive property, HA nanoparticles are usually introduced onto the surface of organic polymeric scaffolds through biomineralization in the field of bone tissue engineering. It has been reported that the HA nanoparticles can modify the surface property of PLA microspheres and promote greatly the cell attachment and proliferation [21]. In addition to the biomineralization, HA can also be nanocomposited into organic polymers by grafting organic polymer chains onto the surface of inorganic HA nanoparticles [22,23]. In order to develop new hemostatic agents with satisfactory properties, this work reported the surface modification and hemostatic property of porous microspheres fabricated by hydroxyapatite nanoparticles-graft-poly(D,L-lactide) (nHA-g-PDLLA) nanocomposites. The surface modification of nanocomposite microspheres was realized through alkaline treatment for the control of pore size and surface property of microspheres. The hemostatic properties of these porous microspheres were evaluated by the platelet adhesion and the whole blood clotting tests. To the best of our knowledge, the porous microspheres fabricated by nHA-gPDLLA nanocomposites were the first attempt for hemostatic applications. The results of this work provide crucial information for developing new porous nanocomposite microspheres with high hemostatic efficacy. 2. Experimental section 2.1. Materials Hydroxyapatite nanoparticles (nHA) were purchased from Aldrich and dried in a vacuum oven at 120 C for 48 h before use. D,L-lactide (DLLA) monomer was purchased from Purac and purified by recrystallization from dry ethyl acetate. Stannous octoate (Sn(Oct)2) was purchased from Sigma and prepared to solution in dried toluene. Toluene was purchased from Beijing Chemical Co. and purified by distillation. Poly(vinyl alcohol) (PVA) with hydrolysis degree of 87e89% and weight average molecular weight (Mw) of 85,000e146,000 was purchased from Aldrich. Other chemical reagents were all of analytical grade and purchased from Beijing Chemical Co. 2.2. Synthesis of nHA-g-PDLLA nanocomposites nHA-g-PDLLA nanocomposites were synthesized by in situ grafting polymerization of DLLA from the surface of HA nanoparticles under argon atmosphere according to our previous work [22,24]. In the present work, nHA-g-PDLLA were designed with HA proportions of 15 wt% and 25 wt% in feed, which were named as S15 and S25, respectively.
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60min and 90min under gentle mechanical stirring, respectively. After that, the specimens were washed with extensive distilled water to remove the remained alkaline. Before hemostasis test, the treated and untreated microspheres were sterilized by g-irradiation under 30 k Gy doses with 60Co as radiation source. 2.4. Characterization of microspheres The morphology of microspheres was examined by scanning electron microscope (SEM) (JEOL JSM-6701F, Japan). The microspheres were mounted on metal stubs with double-sided tape and coated with gold. HA contents in nanocomposite microspheres were measured by a thermogravimetric analyzer (TGA) (TA Instruments, Q500). Samples were heated from 50 C to 650 C at a rate of 20 C/min under N2 atmosphere. The calcium contents on the surface of microspheres were determined by X-ray photoelectron spectroscopy (XPS) analyzer (ESCALab250i-XL electron spectrometer). 2.5. Platelet adhesion [7,13] The platelet rich plasma (PRP) was isolated from human whole blood. The obtained platelet pellets were resuspended into HepesTyrode buffer with a concentration of 3 105/mL. Microspheres were incubated with 2 mL resuspended PRP at 37 C for 60min. To remove the non-adherent platelets, the microspheres were thoroughly washed by phosphate-buffered saline (PBS) without calcium and magnesium. The adherent platelets were lysed with 0.9% Triton-X100 in PBS at 37 C for 60min to release the lactate dehydrogenase (LDH) enzyme. The released LDH enzyme assay was measured by using the LDH/LD Kit (Sigma-Aldrich, USA) according to the manufacturer instructions. 2.6. Whole blood clotting The whole blood clotting tests were performed according to references [8,26]. Microspheres were placed into polypropylene tubes and prewarmed at 37 C. The obtained citrated whole blood (200 mL) was slowly dropped into the tube containing microspheres, and then 20 mL of 0.2 M CaCl2 was added to start the blood clotting. The samples were incubated at 37 C with a slight shake at 30 rpm for 10min. After that, deionized water (25 mL) was carefully transferred into the tube to hemolyze the red blood cells that were not participating in the clotting. The resultant hemoglobin solution was tested by the microplate reader (MULTISCAN MK3, Thermo Electron Corporation) with setting absorbance wavelength at 540 nm. The morphologies of the resultant microspheres were examined by SEM. 3. Results and discussion 3.1. Characterization of microspheres
2.3. Fabrication and structure control of porous nanocomposite microspheres Porous nanocomposite microspheres of S15, S25 and neat PDLLA (S0) were fabricated by a modified double emulsion method (W1/ O/W2), where S0 is selected as a control [25]. For the preparation of S15 and S25 microspheres, the concentration of the organic phase (O), the inner aqueous phase (W1) and the outer aqueous phase (W2) was designed as 6%, 8% and 0.1% respectively. For the preparation of S0 microspheres, the above mentioned three parameters were 4.8%, 6%, and 0.1% respectively. To regulate the structures, microspheres were immersed in 2 M ethylenediamine aqueous solution at room temperature for 30min,
Fig. 1 shows the morphology of the three kinds of porous microspheres before and after alkaline treatment. The obvious changes of porous structure and surface morphology of nHA-gPDLLA nanocomposite microspheres were found after alkaline treatment. For S0, more and larger pores appeared on the surface of microspheres due to the degradation of more PDLLA with increasing alkaline treatment time. For nanocomposite microspheres, in addition to the change of porous structure like S0 sample, HA nanoparticles exposed stably on the surface of porous microspheres were observed, and the amount of exposed HA nanoparticles depended on both alkaline treatment time and the amount of HA nanoparticles in nHA-g-PDLLA nanocomposite
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Fig. 1. SEM images of microspheres fabricated by S0, S15 and S25 after alkaline treatment for 0, 30, 60 and 90min, respectively.
microspheres. Therefore, more HA nanoparticles were visible on the microsphere surface of S25 than that of S15 after alkaline treatment for the same time length. It is also found that the microspheres with higher HA contents show the pores with larger diameter after alkaline treatment. This is due to the weaker stability of primary emulsion (W1/O) with higher content of HA. The results of Fig. 1 indicate that the surface morphology and porous structures of nHA-g-PDLLA nanocomposite microspheres could be well controlled by both alkaline treatment and nanocomposite composition. Fig. 2A and B show the TGA weight loss curves of S15 and S25 microspheres before and after alkaline treatment. Only one thermal decomposition stage with a rapid weight loss around 350 C was found, which was contributed to the decomposition of PDLLA component in the nanocomposite microspheres. The almost unchanged TGA curves at temperatures above 400 C indicated the stability of HA components during heating process. Therefore, based on the TGA curves, the HA and calcium contents of nanocomposite microspheres could be determined according to the weight remain of microspheres, and the results were shown in Fig. 2C and D. As shown in Fig. 2C, the HA contents of original S15 and S25 microspheres were 16.26% and 24.66%, respectively, which are well
in accordance to their contents of nHA-g-PDLLA nanocomposites in feed. The HA contents of S15 or S25 microspheres increased with increasing alkaline treatment time, indicating the degradation of more PDLLA components. According to the following equation, the calcium contents of nanocomposite microspheres were calculated and the results were shown in Fig. 2D.
CaContentðwt%Þ ¼
5 MCa HAContentðwt%Þ MCa5 ðOHÞðPO4 Þ3
The calcium contents of microspheres for S15-0 (where the “0” denotes the alkaline treatment for 0 min), S15e30, S15e60 and S15-90 are 6.48%, 7.19%, 7.38% and 8.53%, respectively, while those for S25e0, S25e30, S25-60, and S25-90 are 9.82%, 10.72%, 12.49%, and 13.06%, respectively. Obviously, the calcium contents of S25 microspheres were higher than those of S15 microspheres, and the calcium contents for S15 or S25 samples increased with increasing the alkaline treatment time. Fig. 3 shows the XPS analysis on nanocomposite microspheres before and after alkaline treatment. For both S15 and S25 microspheres, the intensities of peaks corresponding to Ca2p and P2p increased with alkaline treatment. Because the two peaks are
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Fig. 2. TGA curves of microspheres fabricated by S15 (A) and S25 (B) as well as the calculated HA contents (C) and calcium contents (D).
Fig. 3. X-ray photoelectron spectroscopy of microspheres fabricated by S15 (A) and S25 (B) after alkaline treatment for 0, 30, 60 and 90min.
attributed to the contents of HA nanoparticles on the surface of porous microspheres, the increased peak intensity of Ca2p and P2p indicates that more HA nanoparticles concentrated on the surface of porous microspheres. The results of Fig. 3 also show that both the higher content of HA nanoparticles in nanocomposite microspheres and longer time of alkaline treatment for microspheres lead to the more HA nanoparticles exposed on the surface of porous microspheres. The XPS results were consistent with SEM and TGA results. 3.2. Platelet adhesion Fig. 4 shows the number of platelets adhered on the surface of microspheres fabricated by PDLLA polymer and nHA-g-PDLLA nanocomposites. It was found that the number of platelets adhered onto both S15 and S25 nanocomposite microspheres increased
with increasing alkaline treatment time, whereas the platelet number adhered onto S0 (PDLLA) microspheres was independent on the alkaline treatment time. At the same time node of alkaline treatment, the adhered platelet number onto microspheres was in an order of S0 < S15 < S25. Combined with characterization by TGA, SEM and XPS, the results in Fig. 4 indicated that the analysis of adhered platelet number assay was in accordance with the content of HA nanoparticles exposed on the surface of porous microspheres. Therefore, the inorganic HA nanoparticles and their exposures on the surface play an important role in platelets adhesion onto porous microspheres. 3.3. Whole blood clotting The whole blood clotting was analyzed by the microplate reader
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Fig. 4. Effect of alkaline treatment time and nanocomposition on platelets adhesion onto microspheres after incubation for 60 min (* indicate a significant difference, p < 0.05, analyzed by one way ANOVA, n ¼ 4.)
with setting absorbance wavelength at 540 nm and the results were shown Fig. 5. The lower absorbance value means a faster clotting rate. From Fig. 5A, it was found that the clotting rates of microspheres are in an order of S25 > S15 > S0 at the same time node of alkaline treatment. After alkaline treatment for longer time, the clotting rates of all the three kinds of microspheres increased (Sx-90 > Sx-60 > Sx-30> Sx-0, where x presents the HA proportion in nanocomposites designed as 0, 15 and 25%). For S0 microspheres, the clotting rate after alkaline treatment was a bit higher than that without alkaline treatment, and the resultant clots as shown in Fig. 5B were basically the same. For PDLLA microspheres even without HA, the positive amine groups on the surface induced by ethanediamine solution can attract negatively-charged residues on red blood cells (RBC) membranes [18,27] and promote the interaction with other negative-charged ingredients in blood during the coagulation cascade. For S15 and S25 microspheres, the blood clotting rate dramatically increased compared with that of neat PDLLA microspheres (S0), and the forming clots within these microspheres were more visible, especially for S25 samples as shown in Fig. 5B. These results demonstrate that the content of HA nanoparticles in nHA-g-PDLLA nanocomposite microspheres was an essential factor affecting the hemostatic ability. It has been known that HA has a fierce
adsorption with fibrinogen as well as plasma proteins, which can consequently enhance the platelet aggregation [13,14]. In addition, Ca2þ stemmed from HA can specifically activate the contact pathway because Ca2þ is a prerequisite for activation of prothrombin, coagulation factors VII, IX, and X [16,28]. Fig. 5 also shows that the S25 microspheres have superiority in accelerating whole blood clotting to S15 microspheres after alkaline treatment for the same time length. Based on the results of Fig. 2, it could be concluded that the HA contents within the microspheres play an important role in accelerating the clotting rate. As a result, the thrombin generation and resultant fibrin formation were accelerated on the microspheres with higher HA contents, and the clotting spots seemed to gather and more red blood cells were trapped into the clot, as shown in Fig. 5B. Fig. 6 shows the morphology of microspheres after blood clotting assay. Fibrins formed on microspheres fabricated by only PDLLA without any HA were hard to be seen, while fibrins were much more on microspheres fabricated by nHA-g-PDLLA nanocomposites. Even so for S0-90, some cells are still left even after a hemolyzation progress for the improvement of hydrophilicity by amine groups. For the microspheres fabricated by S15-60, the accumulation trace of clot around the HA nanoparticles could be found as shown by the box in Fig. 6B2. For the microspheres fabricated by S25-90 with much more exposed HA nanoparticles, the formed fibrins totally overlapped on the framework surface of porous microspheres and the formation trace of clot was hard to be distinguished (Fig. 6C). The results in Fig. 6C also shows that the microspheres with higher HA contents are more easily deformed, resulting in the faster clotting formation and better hemostatic effects. In order to check the interaction between HA nanoparticles and blood, the morphology of neat HA nanoparticles before and after dropped with whole blood was shown in Fig. 7. The morphology of neat HA nanoparticles (Fig. 7A) was in accordance with that of exposed HA nanoparticles on nanocomposite microspheres (see Fig. 1, S25 sample after alkaline treatment for 90min). When less blood was added, branched meringue formed and the magnification image shows the visible HA nanoparticles combined within meringue (Fig. 7B). As more blood was added, the meringue gradually converged (Fig. 7C) and finally formed a continuous and thickened layer (Fig. 7D). Also, it was clearly seen that the blood red cells were trapped into clot (Fig. 7C and D). The progress of clot formation can be explained by serials of biochemical reactions which were induced by the huge absorption and energetic activation of HA nanoparticles. Samples in Fig. 6 were edged porous
Fig. 5. Effects of alkaline treatment time and nanocomposition on blood clotting rates (A) and the final forming clots for microspheres after blood clotting assay (B).
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Fig. 6. SEM photographs of microspheres after blood clotting assay. A, B and C are microspheres fabricated by S0-90, S15e60 and S25-90, respectively. The bottoms are enlarged images of uppers.
microspheres after hemolyzing by water from the clot, so seldom blood red cells could be observed. Therefore, based on the above results and discussion, it can be concluded that the HA nanoparticles are the critical factor for hemostatic activity of microspheres fabricated by nHA-g-PDLLA nanocomposites. 3.4. Hemostatic mechanism of porous nanocomposite microspheres
Fig. 7. SEM photographs of commercial HA nanoparticles (A) and these nanoparticles after blood clotting assay with 1 mL (B), 2 mL (C) and 4 mL (D) whole blood, respectively. The rights are enlarged images of lefts.
The alkaline treatment for nHA-g-PDLLA microspheres leads to the hydrolysis of PDLLA chains via ester bond, while HA nanoparticles remain stable. Consequently, the hemostatic mechanism of nanocomposite microspheres was likely to be analyzed through their structure and composition. Alkaline treatment enlarges the original pores of microspheres as shown in Fig. 1, resulting in the larger interaction surface area which is favorable for hemostatic activity. Meanwhile, the exposed HA nanoparticles on the surface of porous microspheres significantly accelerate the blood clotting rate for their intervention in the coagulation cascade. This is due to the adsorption of proteins such as fibrinogen in blood plasma to HA nanoparticles, followed by platelet adhesion, platelet release reaction and platelet aggregation [13]. During the formation of earlier fibrin, the strong adsorption of platelets to HA nanoparticles and the recruitment of red blood cells within the interwoven network of fibrin result in the thrombus enlargement and the final clot formation. The recruited red blood cells not only provide clotting substance but also release phosphatidylserine on the membrane surface to facilitate the activation of platelets [29]. In addition, HA nanoparticles provide high Ca2þ concentration surroundings by releasing Ca2þ which greatly accelerate the coagulation cascade. Specifically, Ca2þ ions promote the secretion named hemostatic mediators of platelets [16,30] and the transformation of fibrinogen to fibrin [16]. Besides, Ca2þ ions mediate many other biochemical bindings and help to maintain the structures and properties of many biochemical intermediate products in the blood coagulation cascade [30e32]. In the present porous nanocomposite microspheres, the component of organic PDLLA copolymer mainly functions as skeleton to provide large surface area for coagulation, while the component of inorganic HA nanoparticles takes effect for hemostatic activity. The higher HA content not only accelerates the blood clotting rate, but also attenuates the mechanical strength of
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Fig. 8. Schematic diagram for the fabrication of porous microspheres with exposed HA nanoparticles and the conjectural blood clotting process of these microspheres.
microspheres. During the coagulation cascade, the accelerating formed thrombus will be loaded to the microspheres. When the thrombus is overloaded, the microspheres are deformed and the thrombus in turn converges to form a bigger clot for continuing the coagulation cascade. According to the explanation above, the hemostatic mechanism of porous microspheres fabricated by nHA-g-PDLLA nanocomposites was proposed, as shown in Fig. 8.
[8]
[9] [10]
[11]
4. Conclusions
[12]
Porous microspheres with remarkable hemostatic efficacy were developed by using the synthetic nHA-g-PDLLA nanocomposites and the subsequent alkaline treatment. These nanocomposite microspheres with HA nanoparticles embedded in microspheres and exposed on the surface can greatly accelerate the blood coagulation cascade and promote the platelets adhesion. During the interaction of blood and nanocomposite microspheres, the HA nanoparticles not only strongly absorb protein constituents in blood, but also provide a high Ca2þ concentration in the surroundings for hemostatic property. The results of this work reveal the potentials of porous microspheres fabricated by nHA-g-PDLLA nanocomposites as effective hemostatic agents both in vitro and in vivo for hemorrhage.
[13]
[14]
[15]
[16] [17]
[18] [19]
Acknowledgements
[20]
This study is supported by the National Natural Science Foundation of China (Grant no 51025314).
[21]
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Structural control and hemostatic properties of porous microspheres fabricated by hydroxyapatite-graft-poly(D,L-lactide) nanocomposites Leyuan Song a, Lei Sun b, Ni Jiang a, *, Zhihua Gan a, ** a State Key Laboratory of Organic-Inorganic Composites, Beijing Laboratory of Biomedical Materials, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China b Beijing Institute of Traumatology and Orthopaedics, Jishuitan Hospital, Beijing 100035, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2016 Received in revised form 31 August 2016 Accepted 1 September 2016 Available online 1 September 2016
Nanocomposite microspheres with tunable porous structures were studied in this work as hemostatic agents. Hydroxyapatite nanoparticles-graft-poly(D,L-lactide) (nHA-g-PDLLA) nanocomposites were synthesized first and then fabricated into porous microspheres by a modified double emulsion solvent evaporation technique. Alkaline treatment were carried out on nanocomposite microspheres for the exposure of HA nanoparticles on the surface of porous microspheres. The porous structure and nanocomposition endue the nHA-g-PDLLA microspheres with huge specific surface area and excellent interfaces for blood clotting. This work reveals the great potentials of nHA-g-PDLLA nanocomposite microspheres as hemostatic agents. © 2016 Published by Elsevier Ltd.
Keywords: Polymer-matrix composites (PMCs) Interface Surface treatments
1. Introduction Uncontrolled massive hemorrhaging has always been the leading cause of deaths after injury whether in war or daily life [1e3]. In fact, natural response to injury of human body can deal with light wound with three stages, i.e., the formation of a platelet plug, an enzymatic cascade resulting in the formation of fibrin, and the dissolution of clot and healing of wound site [4,5]. Therefore, tremendous hemostatic agents have been developed to intervene in the coagulation cascade for arresting bleeding and stabilizing wound. Current hemostatic agents approved clinically act through passive, active or a combination of mechanisms [6]. For example, oxidized/oxidized regenerated cellulose, gelatin sponges, collagen pads and sponges play the passive hemostatic roles through surface acting, while thrombin, fibrin sealants and hemostatic patches are active ones that partake in the coagulation cascade by providing biologically active components. Great efforts have been made to develop novel composite hemostatic agents that combined passive and active components. Polyphosphate refined the chitosan dressings, calcium-modified microporous starch, and graphene contained chitosan-poly(vinyl alcohol) nanofibers have been
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (N. Jiang), [email protected] (Z. Gan). http://dx.doi.org/10.1016/j.compscitech.2016.09.001 0266-3538/© 2016 Published by Elsevier Ltd.
reported to be potent wound dressings or beneficial for wound healing [7e9]. Hydroxyapatite (HA, Ca10(PO4)6(OH)2), which is the main inorganic component of natural bone, has been widely studied in bone tissue engineering and industrial catalysts [10,11]. It has been reported that HA nanoparticles possess an extraordinary adsorption of proteins due to the surface charge and particle texture [12]. The easy protein-binding surface of biomaterials is favorable for the platelets adhesion and the blood clotting formation due to the adsorbed proteins in blood coagulation cascade [13]. Moreover, HA 2þ exhibits both acid (PO3 ion) active sites based 4 ion) and base (Ca 2þ on its crystal structure [14,15]. The Ca is an important factor in blood coagulation cascade, named the fourth clotting factor which is also the only inorganic factor [16,17]. It has been reported that the addition of Ca2þ ions not only decreases the induction period of the fibrinogen-fibrin system in the coagulation cascade, but also greatly promotes the fibrin monomer polymerization [18]. Therefore, the calcium-modified microporous starch prepared by oxidization and self-assembly with Ca2þ [8] has been reported to own potent hemostatic efficiency for hemorrhage control because of the acceleration of Ca2þ for blood clotting. Microspheres fabricated by synthetic biodegradable polymers such as polylactide (PLA), its copolymer with polyglycolide (PLGA), and poly(ε-caprolactone) (PCL) are widely studied for tissue engineering and drug delivery. The microspheres are usually prepared by emulsion solvent evaporation techniques [19]. However, because
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of the lack of bioactive sites and the intense hydrophobicity, great efforts have been made in fabricating composite microspheres to meet the versatile requirements in application [20]. In consideration of the bioactive property, HA nanoparticles are usually introduced onto the surface of organic polymeric scaffolds through biomineralization in the field of bone tissue engineering. It has been reported that the HA nanoparticles can modify the surface property of PLA microspheres and promote greatly the cell attachment and proliferation [21]. In addition to the biomineralization, HA can also be nanocomposited into organic polymers by grafting organic polymer chains onto the surface of inorganic HA nanoparticles [22,23]. In order to develop new hemostatic agents with satisfactory properties, this work reported the surface modification and hemostatic property of porous microspheres fabricated by hydroxyapatite nanoparticles-graft-poly(D,L-lactide) (nHA-g-PDLLA) nanocomposites. The surface modification of nanocomposite microspheres was realized through alkaline treatment for the control of pore size and surface property of microspheres. The hemostatic properties of these porous microspheres were evaluated by the platelet adhesion and the whole blood clotting tests. To the best of our knowledge, the porous microspheres fabricated by nHA-gPDLLA nanocomposites were the first attempt for hemostatic applications. The results of this work provide crucial information for developing new porous nanocomposite microspheres with high hemostatic efficacy. 2. Experimental section 2.1. Materials Hydroxyapatite nanoparticles (nHA) were purchased from Aldrich and dried in a vacuum oven at 120 C for 48 h before use. D,L-lactide (DLLA) monomer was purchased from Purac and purified by recrystallization from dry ethyl acetate. Stannous octoate (Sn(Oct)2) was purchased from Sigma and prepared to solution in dried toluene. Toluene was purchased from Beijing Chemical Co. and purified by distillation. Poly(vinyl alcohol) (PVA) with hydrolysis degree of 87e89% and weight average molecular weight (Mw) of 85,000e146,000 was purchased from Aldrich. Other chemical reagents were all of analytical grade and purchased from Beijing Chemical Co. 2.2. Synthesis of nHA-g-PDLLA nanocomposites nHA-g-PDLLA nanocomposites were synthesized by in situ grafting polymerization of DLLA from the surface of HA nanoparticles under argon atmosphere according to our previous work [22,24]. In the present work, nHA-g-PDLLA were designed with HA proportions of 15 wt% and 25 wt% in feed, which were named as S15 and S25, respectively.
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60min and 90min under gentle mechanical stirring, respectively. After that, the specimens were washed with extensive distilled water to remove the remained alkaline. Before hemostasis test, the treated and untreated microspheres were sterilized by g-irradiation under 30 k Gy doses with 60Co as radiation source. 2.4. Characterization of microspheres The morphology of microspheres was examined by scanning electron microscope (SEM) (JEOL JSM-6701F, Japan). The microspheres were mounted on metal stubs with double-sided tape and coated with gold. HA contents in nanocomposite microspheres were measured by a thermogravimetric analyzer (TGA) (TA Instruments, Q500). Samples were heated from 50 C to 650 C at a rate of 20 C/min under N2 atmosphere. The calcium contents on the surface of microspheres were determined by X-ray photoelectron spectroscopy (XPS) analyzer (ESCALab250i-XL electron spectrometer). 2.5. Platelet adhesion [7,13] The platelet rich plasma (PRP) was isolated from human whole blood. The obtained platelet pellets were resuspended into HepesTyrode buffer with a concentration of 3 105/mL. Microspheres were incubated with 2 mL resuspended PRP at 37 C for 60min. To remove the non-adherent platelets, the microspheres were thoroughly washed by phosphate-buffered saline (PBS) without calcium and magnesium. The adherent platelets were lysed with 0.9% Triton-X100 in PBS at 37 C for 60min to release the lactate dehydrogenase (LDH) enzyme. The released LDH enzyme assay was measured by using the LDH/LD Kit (Sigma-Aldrich, USA) according to the manufacturer instructions. 2.6. Whole blood clotting The whole blood clotting tests were performed according to references [8,26]. Microspheres were placed into polypropylene tubes and prewarmed at 37 C. The obtained citrated whole blood (200 mL) was slowly dropped into the tube containing microspheres, and then 20 mL of 0.2 M CaCl2 was added to start the blood clotting. The samples were incubated at 37 C with a slight shake at 30 rpm for 10min. After that, deionized water (25 mL) was carefully transferred into the tube to hemolyze the red blood cells that were not participating in the clotting. The resultant hemoglobin solution was tested by the microplate reader (MULTISCAN MK3, Thermo Electron Corporation) with setting absorbance wavelength at 540 nm. The morphologies of the resultant microspheres were examined by SEM. 3. Results and discussion 3.1. Characterization of microspheres
2.3. Fabrication and structure control of porous nanocomposite microspheres Porous nanocomposite microspheres of S15, S25 and neat PDLLA (S0) were fabricated by a modified double emulsion method (W1/ O/W2), where S0 is selected as a control [25]. For the preparation of S15 and S25 microspheres, the concentration of the organic phase (O), the inner aqueous phase (W1) and the outer aqueous phase (W2) was designed as 6%, 8% and 0.1% respectively. For the preparation of S0 microspheres, the above mentioned three parameters were 4.8%, 6%, and 0.1% respectively. To regulate the structures, microspheres were immersed in 2 M ethylenediamine aqueous solution at room temperature for 30min,
Fig. 1 shows the morphology of the three kinds of porous microspheres before and after alkaline treatment. The obvious changes of porous structure and surface morphology of nHA-gPDLLA nanocomposite microspheres were found after alkaline treatment. For S0, more and larger pores appeared on the surface of microspheres due to the degradation of more PDLLA with increasing alkaline treatment time. For nanocomposite microspheres, in addition to the change of porous structure like S0 sample, HA nanoparticles exposed stably on the surface of porous microspheres were observed, and the amount of exposed HA nanoparticles depended on both alkaline treatment time and the amount of HA nanoparticles in nHA-g-PDLLA nanocomposite
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Fig. 1. SEM images of microspheres fabricated by S0, S15 and S25 after alkaline treatment for 0, 30, 60 and 90min, respectively.
microspheres. Therefore, more HA nanoparticles were visible on the microsphere surface of S25 than that of S15 after alkaline treatment for the same time length. It is also found that the microspheres with higher HA contents show the pores with larger diameter after alkaline treatment. This is due to the weaker stability of primary emulsion (W1/O) with higher content of HA. The results of Fig. 1 indicate that the surface morphology and porous structures of nHA-g-PDLLA nanocomposite microspheres could be well controlled by both alkaline treatment and nanocomposite composition. Fig. 2A and B show the TGA weight loss curves of S15 and S25 microspheres before and after alkaline treatment. Only one thermal decomposition stage with a rapid weight loss around 350 C was found, which was contributed to the decomposition of PDLLA component in the nanocomposite microspheres. The almost unchanged TGA curves at temperatures above 400 C indicated the stability of HA components during heating process. Therefore, based on the TGA curves, the HA and calcium contents of nanocomposite microspheres could be determined according to the weight remain of microspheres, and the results were shown in Fig. 2C and D. As shown in Fig. 2C, the HA contents of original S15 and S25 microspheres were 16.26% and 24.66%, respectively, which are well
in accordance to their contents of nHA-g-PDLLA nanocomposites in feed. The HA contents of S15 or S25 microspheres increased with increasing alkaline treatment time, indicating the degradation of more PDLLA components. According to the following equation, the calcium contents of nanocomposite microspheres were calculated and the results were shown in Fig. 2D.
CaContentðwt%Þ ¼
5 MCa HAContentðwt%Þ MCa5 ðOHÞðPO4 Þ3
The calcium contents of microspheres for S15-0 (where the “0” denotes the alkaline treatment for 0 min), S15e30, S15e60 and S15-90 are 6.48%, 7.19%, 7.38% and 8.53%, respectively, while those for S25e0, S25e30, S25-60, and S25-90 are 9.82%, 10.72%, 12.49%, and 13.06%, respectively. Obviously, the calcium contents of S25 microspheres were higher than those of S15 microspheres, and the calcium contents for S15 or S25 samples increased with increasing the alkaline treatment time. Fig. 3 shows the XPS analysis on nanocomposite microspheres before and after alkaline treatment. For both S15 and S25 microspheres, the intensities of peaks corresponding to Ca2p and P2p increased with alkaline treatment. Because the two peaks are
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Fig. 2. TGA curves of microspheres fabricated by S15 (A) and S25 (B) as well as the calculated HA contents (C) and calcium contents (D).
Fig. 3. X-ray photoelectron spectroscopy of microspheres fabricated by S15 (A) and S25 (B) after alkaline treatment for 0, 30, 60 and 90min.
attributed to the contents of HA nanoparticles on the surface of porous microspheres, the increased peak intensity of Ca2p and P2p indicates that more HA nanoparticles concentrated on the surface of porous microspheres. The results of Fig. 3 also show that both the higher content of HA nanoparticles in nanocomposite microspheres and longer time of alkaline treatment for microspheres lead to the more HA nanoparticles exposed on the surface of porous microspheres. The XPS results were consistent with SEM and TGA results. 3.2. Platelet adhesion Fig. 4 shows the number of platelets adhered on the surface of microspheres fabricated by PDLLA polymer and nHA-g-PDLLA nanocomposites. It was found that the number of platelets adhered onto both S15 and S25 nanocomposite microspheres increased
with increasing alkaline treatment time, whereas the platelet number adhered onto S0 (PDLLA) microspheres was independent on the alkaline treatment time. At the same time node of alkaline treatment, the adhered platelet number onto microspheres was in an order of S0 < S15 < S25. Combined with characterization by TGA, SEM and XPS, the results in Fig. 4 indicated that the analysis of adhered platelet number assay was in accordance with the content of HA nanoparticles exposed on the surface of porous microspheres. Therefore, the inorganic HA nanoparticles and their exposures on the surface play an important role in platelets adhesion onto porous microspheres. 3.3. Whole blood clotting The whole blood clotting was analyzed by the microplate reader
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Fig. 4. Effect of alkaline treatment time and nanocomposition on platelets adhesion onto microspheres after incubation for 60 min (* indicate a significant difference, p < 0.05, analyzed by one way ANOVA, n ¼ 4.)
with setting absorbance wavelength at 540 nm and the results were shown Fig. 5. The lower absorbance value means a faster clotting rate. From Fig. 5A, it was found that the clotting rates of microspheres are in an order of S25 > S15 > S0 at the same time node of alkaline treatment. After alkaline treatment for longer time, the clotting rates of all the three kinds of microspheres increased (Sx-90 > Sx-60 > Sx-30> Sx-0, where x presents the HA proportion in nanocomposites designed as 0, 15 and 25%). For S0 microspheres, the clotting rate after alkaline treatment was a bit higher than that without alkaline treatment, and the resultant clots as shown in Fig. 5B were basically the same. For PDLLA microspheres even without HA, the positive amine groups on the surface induced by ethanediamine solution can attract negatively-charged residues on red blood cells (RBC) membranes [18,27] and promote the interaction with other negative-charged ingredients in blood during the coagulation cascade. For S15 and S25 microspheres, the blood clotting rate dramatically increased compared with that of neat PDLLA microspheres (S0), and the forming clots within these microspheres were more visible, especially for S25 samples as shown in Fig. 5B. These results demonstrate that the content of HA nanoparticles in nHA-g-PDLLA nanocomposite microspheres was an essential factor affecting the hemostatic ability. It has been known that HA has a fierce
adsorption with fibrinogen as well as plasma proteins, which can consequently enhance the platelet aggregation [13,14]. In addition, Ca2þ stemmed from HA can specifically activate the contact pathway because Ca2þ is a prerequisite for activation of prothrombin, coagulation factors VII, IX, and X [16,28]. Fig. 5 also shows that the S25 microspheres have superiority in accelerating whole blood clotting to S15 microspheres after alkaline treatment for the same time length. Based on the results of Fig. 2, it could be concluded that the HA contents within the microspheres play an important role in accelerating the clotting rate. As a result, the thrombin generation and resultant fibrin formation were accelerated on the microspheres with higher HA contents, and the clotting spots seemed to gather and more red blood cells were trapped into the clot, as shown in Fig. 5B. Fig. 6 shows the morphology of microspheres after blood clotting assay. Fibrins formed on microspheres fabricated by only PDLLA without any HA were hard to be seen, while fibrins were much more on microspheres fabricated by nHA-g-PDLLA nanocomposites. Even so for S0-90, some cells are still left even after a hemolyzation progress for the improvement of hydrophilicity by amine groups. For the microspheres fabricated by S15-60, the accumulation trace of clot around the HA nanoparticles could be found as shown by the box in Fig. 6B2. For the microspheres fabricated by S25-90 with much more exposed HA nanoparticles, the formed fibrins totally overlapped on the framework surface of porous microspheres and the formation trace of clot was hard to be distinguished (Fig. 6C). The results in Fig. 6C also shows that the microspheres with higher HA contents are more easily deformed, resulting in the faster clotting formation and better hemostatic effects. In order to check the interaction between HA nanoparticles and blood, the morphology of neat HA nanoparticles before and after dropped with whole blood was shown in Fig. 7. The morphology of neat HA nanoparticles (Fig. 7A) was in accordance with that of exposed HA nanoparticles on nanocomposite microspheres (see Fig. 1, S25 sample after alkaline treatment for 90min). When less blood was added, branched meringue formed and the magnification image shows the visible HA nanoparticles combined within meringue (Fig. 7B). As more blood was added, the meringue gradually converged (Fig. 7C) and finally formed a continuous and thickened layer (Fig. 7D). Also, it was clearly seen that the blood red cells were trapped into clot (Fig. 7C and D). The progress of clot formation can be explained by serials of biochemical reactions which were induced by the huge absorption and energetic activation of HA nanoparticles. Samples in Fig. 6 were edged porous
Fig. 5. Effects of alkaline treatment time and nanocomposition on blood clotting rates (A) and the final forming clots for microspheres after blood clotting assay (B).
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Fig. 6. SEM photographs of microspheres after blood clotting assay. A, B and C are microspheres fabricated by S0-90, S15e60 and S25-90, respectively. The bottoms are enlarged images of uppers.
microspheres after hemolyzing by water from the clot, so seldom blood red cells could be observed. Therefore, based on the above results and discussion, it can be concluded that the HA nanoparticles are the critical factor for hemostatic activity of microspheres fabricated by nHA-g-PDLLA nanocomposites. 3.4. Hemostatic mechanism of porous nanocomposite microspheres
Fig. 7. SEM photographs of commercial HA nanoparticles (A) and these nanoparticles after blood clotting assay with 1 mL (B), 2 mL (C) and 4 mL (D) whole blood, respectively. The rights are enlarged images of lefts.
The alkaline treatment for nHA-g-PDLLA microspheres leads to the hydrolysis of PDLLA chains via ester bond, while HA nanoparticles remain stable. Consequently, the hemostatic mechanism of nanocomposite microspheres was likely to be analyzed through their structure and composition. Alkaline treatment enlarges the original pores of microspheres as shown in Fig. 1, resulting in the larger interaction surface area which is favorable for hemostatic activity. Meanwhile, the exposed HA nanoparticles on the surface of porous microspheres significantly accelerate the blood clotting rate for their intervention in the coagulation cascade. This is due to the adsorption of proteins such as fibrinogen in blood plasma to HA nanoparticles, followed by platelet adhesion, platelet release reaction and platelet aggregation [13]. During the formation of earlier fibrin, the strong adsorption of platelets to HA nanoparticles and the recruitment of red blood cells within the interwoven network of fibrin result in the thrombus enlargement and the final clot formation. The recruited red blood cells not only provide clotting substance but also release phosphatidylserine on the membrane surface to facilitate the activation of platelets [29]. In addition, HA nanoparticles provide high Ca2þ concentration surroundings by releasing Ca2þ which greatly accelerate the coagulation cascade. Specifically, Ca2þ ions promote the secretion named hemostatic mediators of platelets [16,30] and the transformation of fibrinogen to fibrin [16]. Besides, Ca2þ ions mediate many other biochemical bindings and help to maintain the structures and properties of many biochemical intermediate products in the blood coagulation cascade [30e32]. In the present porous nanocomposite microspheres, the component of organic PDLLA copolymer mainly functions as skeleton to provide large surface area for coagulation, while the component of inorganic HA nanoparticles takes effect for hemostatic activity. The higher HA content not only accelerates the blood clotting rate, but also attenuates the mechanical strength of
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Fig. 8. Schematic diagram for the fabrication of porous microspheres with exposed HA nanoparticles and the conjectural blood clotting process of these microspheres.
microspheres. During the coagulation cascade, the accelerating formed thrombus will be loaded to the microspheres. When the thrombus is overloaded, the microspheres are deformed and the thrombus in turn converges to form a bigger clot for continuing the coagulation cascade. According to the explanation above, the hemostatic mechanism of porous microspheres fabricated by nHA-g-PDLLA nanocomposites was proposed, as shown in Fig. 8.
[8]
[9] [10]
[11]
4. Conclusions
[12]
Porous microspheres with remarkable hemostatic efficacy were developed by using the synthetic nHA-g-PDLLA nanocomposites and the subsequent alkaline treatment. These nanocomposite microspheres with HA nanoparticles embedded in microspheres and exposed on the surface can greatly accelerate the blood coagulation cascade and promote the platelets adhesion. During the interaction of blood and nanocomposite microspheres, the HA nanoparticles not only strongly absorb protein constituents in blood, but also provide a high Ca2þ concentration in the surroundings for hemostatic property. The results of this work reveal the potentials of porous microspheres fabricated by nHA-g-PDLLA nanocomposites as effective hemostatic agents both in vitro and in vivo for hemorrhage.
[13]
[14]
[15]
[16] [17]
[18] [19]
Acknowledgements
[20]
This study is supported by the National Natural Science Foundation of China (Grant no 51025314).
[21]
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