How to cite this article: Otsuka M, Shikamura M, Otsuka K, Sogo Y, Ito A. 2013. Therapeutic effect of zinc-containing calcium phosphate suspension injection in thermal burn-rats. J Biomed Mater Res Part A 2013:101A:1518–1524.
Trace elements, such as iron, iodine, chromium, copper, zinc, and selenium, are essential to many animal species.1 Severe trace element imbalances can be considered risk factors for several diseases. Deficiencies of iron and iodine cause anemia and hypothyroidism. Fifty micrograms to 20 mg per day of those elements are necessary, because the elements serve as structural or catalytic components of large molecules in tissues. Zinc (Zn) is a cofactor for more than 200 enzymes and present in nearly every cell type in the body.2 Because Zn is known to be essential for all highly protein synthesis, proliferating, and differentiation cells in the human body, Zn promotes the formation of tissues and organs.
As bone has higher Zn concentrations than any other tissue, bone generation is stimulated by Zn ions. However, high Zn concentrations can have serious side effects on cells.3 Therefore, the range of Zn concentrations for an optimal biological response, or therapeutic index, would be very narrow. Kawamura et al.4, 5 used Zn-containing calcium phosphate ceramics as novel inorganic biomaterials in rabbit femora. They reported that the Zn-containing calcium phosphate ceramics promoted the proliferation of MC3T3-E1 cells without a cytotoxic effect up to a zinc content of 1.20 wt %, releasing zinc up to a concentration of 3.53 mg/L. However, the viewpoint of cytotoxicity, a zinc content of 1.20 wt % was also the upper limit for the implantable ceramics. Over a zinc content of 1.20 wt %, the ceramics were cytotoxic owing to elevated zinc release from the device. The slow release of Zn from the ceramics stimulated bone cells and induced bone generation.4, 5 Since then, an injectable suspension containing β-tricalcium phosphate (TCP) powder with Zn has been developed and applied to an animal model of osteoporosis where it was effective at recovering bone mineral density.6–8
In contrast, Zn also has an important role in the healing of thermal burns, immune system functioning, and maintaining. Ibs and Rink9 reported that a variety of in vivo and in vitro effects of zinc on immune cells mainly depend on the zinc concentration. All kinds of immune cells, such as natural killer cells, T cells, and B cells show decreased function after zinc depletion. The impaired immune functions due to zinc deficiency are shown to be reversed by an adequate zinc supplementation. In contrast, excessed Zn concentration by high dosages of Zn also evoke negative effects on immune cells and a direct chemotactic activation of neutrophil granulocytes, and those phenomena are similar to those observed with Zn deficiency. In optimized Zn concentration, Zn suppresses natural killer cell killing and T-cell functions whereas monocytes are activated directly. Optimized Zn concentration by controlled Zn release might, therefore, benefit the healing of thermal burns thorough controlling of immune function to prevent inflammation.
In this article, we applied suspension injections of the candidate Zn-related materials for therapeutic treating thermal burns of Zn-deficient rats, and evaluated hearing process as the first step. Furthermore, to improve the quality of life of the patients during therapeutic treatment, we considered that their dosage forms of the candidate compounds changed to transdermal therapeutic dosage form to avoid injection into injury sites as future purpose.
MATERIALS AND METHODS
A powder of β-TCP containing 10 mol % Zn (β-ZnTCP, 6.17% (w/w) Zn, [Ca2.7Zn0.3(PO4)2]) was prepared as described previously.4, 10 Starting materials were high-purity calcium carbonate (99.99 wt %; Ube Materials, Ube, Japan), reagent-grade phosphoric acid (85 wt %; Nakalai Tesque, Kyoto, Japan), and reagent-grade zinc nitrate hexahydrate (98 wt %; Kanto Chemical Co., Tokyo, Japan). The calcium carbonate was heated at 1000°C for 3 h to obtain calcium oxide. The calcium oxide was added to ultrapure water to obtain a suspension of calcium hydroxide (0.6 Ca mol/L). The suspension was mixed with a zinc nitrate hexahydrate solution (0.5 Zn mol/L) at a molar ratio of 0.10 in Zn/(Ca + Zn). The suspension was vigorously stirred at room temperature by bubbling with nitrogen gas. A phosphoric acid solution (0.6 P mol/L) was added dropwise to the suspensions to produce a gelatinous precipitate. The pH was maintained at 6.5 for several hours by adding a 3% ammonia solution. The reaction mixture was stirred for several days. The precipitate was filtered, dried at 100°C and calcined at 850°C for 3 h followed by pulverization. A powder of α-TCP containing 0.9 mol % Zn (α-ZnTCP, 0.55% (w/w) Zn, [Ca2.973Zn0.027(PO4)2]) was prepared by mixing the β-ZnTCP and pure β-TCP powders at a weight ratio of 11:109, and heating at 1450°C for 5 h followed by pulverization. The heat treatment and pulverization was repeated twice. The β-ZnTCP and α-ZnTCP powders were ground in a high-purity alumina mortar and sieved to a size smaller than 38 μm. Sodium alginate and zinc sulfate hepta-hydrates (ZnSO4) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Other reagents were of analytical grade and used without further purification. All suspension injections were obtained by mixing the powders of ZnSO4, α-ZnTCP, and β-ZnTCP with 2.0 mL of 1% sodium alginate saline solution. The content of Zn in each suspension injections was adjusted to 2.0 mg Zn per animal experiment.
Characterization of calcium phosphates
α-ZnTCP and β-ZnTCP were characterized by powder X-ray diffraction (XRD) and specific surface area measurements. Powder XRD patterns were recorded using a RINT Ultima 3 diffractometer (Rigaku, Tokyo, Japan, conditions; target: Cu, filter: Ni, voltage: 40 kV, current: 40 mA, and receiving slit: 0.1 mm). The specific surface area of dried samples was measured by the nitrogen adsorption method with a surface measurement apparatus (Monosorb, Quantachrome Corporation, Boynton Beach, FL). The powders (about 1.0 g) were dried and degassed in a vacuum at 80°C for at least 1 h in glass sample cells before measurements. Specific surface area (m2/g) was calculated three times for each powder by the single-point BET method.
In vitro Zn release test
The amount of Zn released from the α-Zn-TCP and β-Zn-TCP powders was measured as follows: simulated body fluid6 (SBF) comprised of 142 mM Na+, 5.0 mM K+, 1.5 mM Mg2+, 147.8 mM Cl−, 2.5 mM Ca2+, 4.2 mM HCO3−, 0.5 mM SO42−, and 1.0 mM HPO42+, (pH 7.25) was used as the dissolution medium. Samples (10 mg) and 10 mL of SBF containing 10 mg/100mL Ca2+ were introduced into 50-mL test tubes with a cap. Each tube was fixed on a sample holder in a thermostatically regulated water bath maintained at 37.0 ± 0.1°C, and shaken at 90 rpm. During the release tests, the entire dissolution medium was replaced with 5.0 mL of fresh buffer at various intervals.
Zn concentrations were determined at 213.8 nm by atomic absorption spectrometry (type 180-70, Hitachi Co. Ind., Tokyo, Japan). The in vitro data shown represent the averages of three measurements.
Animal experiments (in vivo test)
All animal experiments and maintenance were performed under conditions approved by the Animal Research Committee of Kobe Pharmaceutical University. Seven-week-old male Whister rats around 120 g in weight purchased from Japan SLC, Shizuoka, Japan were used. The rats were maintained in a light (12 h light/dark) and temperature-controlled (25 ± 2°C) barrier facility throughout the study. The rats were randomly assigned to four groups: C, D1, D2, and D3 (four rats per group). All rats were fed a Zn-deficient diet (shown in Table I) for 1 week. The Zn-deficient and normal diets were supplied by Clea Co. (Tokyo, Japan). The hair on the back of the rats was removed under anesthesia by inhalation of ether, and the burn treatment was performed by pushing a glass bottle (with a flat bottom, 10 mm in diameter and 20 mL in volume) containing hot paraffin at 100°C for 10 s On starting the injection study after the burns treatment for 3 h, the rats in groups D2 and D3 were subcutaneous (s.c.) injected twice at upper and lower sites 5 mm distance from the edge of burns into the back with 2 mL volume of the suspensions containing α-ZnTCP or β-ZnTCP powders by using a needle (No. 26 gage), respectively, whereas the rats in group D1 were injected at upper and lower sites 5 mm distance from the edge of burns with 2 mL volume of the ZnSO4 solution divided 14 times for 14 days, because one time injection of the ZnSO4 solution induced necrosis on the injection sites. After the injection, the wounded site was cleaned with a 70% alcoholic solution, covered with sterilized cotton cloth wetted with the 70% alcoholic solution and then, fixed using flexible tape. The rats in groups D1, D2, and D3 were assigned to receive the Zn-deficient diet after starting the injection study for 3 weeks. After the burns treatment, the rats in group C were assigned to receive an injection of saline with 1% sodium alginate, and given a normal diet. Blood samples were obtained at predetermined time intervals from a tail vein under anesthesia. A rat in group C was scarified by another rat during experiments, so the number of rats in group C was reduced to three.
Table I. Zn-Deficient Diet Used
Vitamin D (IU/100 g)
Ca (mg/100 g)
Zn (mg/100 g)
The data in the table are from Clea Co. (Japan) and Oriental yeast Co. (Japan).
Vit. D and Zn-deficient diet
Evaluation of therapeutic scores of thermal burns
The thermal burns were photographed with a digital camera, and the scab was assigned a therapeutic score by imaging computer software for Windows (Image J, National Institute of Health).
Measurements of plasma alkaline phosphatase activity
Plasma alkaline phosphatase (ALP) activity was determined using the phenyl phosphoric acid method11 with a UV/VIS spectrometer (type UV160, Shimadzu Co. Ind., Kyoto, Japan) at 500 nm. The kits used were supplied by Wako Chem. Co, Tokyo, Japan. The in vivo data shown represent the average of four measurements each
All data are the mean and standard deviation of 3–6 measurements. The statistical analysis was conducted using analysis of variance followed by Tukey's post hoc tests, and p-values of 0.005–0.05 were considered significant.
Characterization of materials
Figure 1 shows the powder XRD profiles for α-ZnTCP and β-ZnTCP. α-ZnTCP had significant diffraction peaks at 2θ = 12.3°, 23.8°, and 30.8° corresponding to those for α-TCP with a high crystallinity. β-ZnTCP had significant diffraction peaks at 2θ = 13.5°, 28.1°, and 31.5° corresponding to those for β-TCP with a high crystallinity although the peak positions are slightly shifted toward a higher angle, because it contained 10 mol % Zn as reported previously.4
Figure 2 shows SEM photographs of α-ZnTCP and β-ZnTCP. The primary particles of β-ZnTCP were less than 1 μm in diameter, and those were aggregated forming secondary particles around 50 μm in diameter with a jagged surface. In contrast, the α-ZnTCP particles had a smooth surface with many dimples, and were around 40–50 μm in diameter. Specific surface areas of α-ZnTCP and β-ZnTCP were 0.303 and 6.37 m2/g, respectively, and the result was consisted with the SEM observations.
In vitro Zn release from ZnTCPs in SBF
Figure 3 shows the in vitro Zn release profiles for the Zn-related powders in SBF. The release of ZnSO4 was very fast at the initial stage, with over 90% of Zn dissolved within 2 h, suggesting that ZnSO4 was highly soluble in water. In contrast, the amounts of Zn released from α-ZnTCP and β-ZnTCP were around 25% and 5% after 24 h. The order of dissolution rates for the Zn-related materials was ZnSO4 > α-ZnTCP > β-ZnTCP.
Effect of crystalline structure of ZnTCP suspension on in vivo plasma Zn levels
To investigate the therapeutic effect of in vivo Zn release on the healing of thermal burns, ZnTCP suspensions were injected into the rats. Figure 4 (left) shows plasma Zn concentration profiles in the rats with thermal burns and healthy (control) rats measured using atomic absorption. The plasma Zn levels in the ZnSO4-administrered model (group D1) increased rapidly and reached 2.8 μg/mL on day 6. Thereafter, they decreased, to around 1.7 μg/mL on days 8–15 and then to around 0.5 μg/mL on day 21. In contrast, the plasma Zn levels in the α-TCP-administered model (group D2) increased to 1.7 μg/mL on day 5, then decreased to less than 0.5 mg/mL after 8 days.
The plasma Zn levels in the β-TCP-administered model (group D3) were to almost the constant values between 0.5 and 1.2 μg/mL on days 3–7, and then decreased to less than 0.5 mg/mL after 8 days. The levels in the nonadministered model (group C) were to almost the same profiles of the group D3, and those were the constant values between 0.5 and 1.2 μg/mL on days 3–7, and then decreased to less than 0.5 mg/mL after 8 days.
The area under the curve for the plasma Zn concentration (Zn-AUC) after 21 days is summarized in Figure 4 (right). The Zn-AUC was significantly higher for group D1 than groups D2, D3, and C. The Zn-AUC of group D1 was the highest, and the order was groups D1 > D2 ≥ D3 ≥ C. However, the Zn-AUC for groups D2 and D3 were not significantly different from that of group C. These results suggested that the ZnSO4 solution increased, but the α-ZnTCP and β-ZnTCP suspensions did not affect, plasma Zn levels.
Effect of the crystalline structure of ZnTCP suspension on alkaline phosphatase activity
Because plasma ALP activity is an important parameter of the healing process, it was measured in the rats with thermal burns.12 Figure 5 (left) shows that before the thermal treatment, the ALP level in the control group (group C) was around 28 IU/L, but after the treatment it fluctuated between 20 and 40 IU/L depending on the conditions. The ALP level of group D1 and D2 rats (ZnSO4 and α-ZnTCP) were higher than that of the control group (group C), and showed a maximum of 48 IU/L at 4 days and 46 IU/L at 5 days, respectively. In contrast, the ALP levels of group D3 (β-ZnTCP administration) were almost the same as those of the control rats (group C), except for 47 IU/L at 21 days.
The area under the curve for ALP (ALP-AUC) after 21 days is summarized in Figure 5 (right). The ALP-AUC was significantly higher for group D1 than groups D1, D2, and C. The ALP-AUC of group D1 was the highest, and the order was groups D1 > D2 = D3 ≥ C. However, the ALP-AUC for groups D2 and D3 were not significantly different from that of group C. These results suggested that the ZnSO4 solution activated all body cells, but the α-ZnTCP and β-ZnTCP suspensions did not.
Effect of the crystalline structure of ZnTCP suspension on healing of thermal burns
Figure 6 shows the healing of wounded skin (the D2 group) after the α-ZnTCP injection. After thermal treatment, the area was deformed and discolored. After 1 day, it formed a blister, the area was drying and then after 4 days it formed a sable. The sable decreased in area with time, and eventually dropped off after 16 days.
To quantitatively evaluate the healing process, the wounded area (Aw) of rat skin was measured using an imaging analyzer. Figure 7 shows the Aw profiles of the thermal burns model after the s.c. administration of various Zn-related material suspensions. The Aw of group C increased about 30% for 2 days, then, decreased with time. The Aw on day 10 and 20 was about 75% and 10%, respectively. The Aw of group D1 given ZnSO4 had almost the same profile as that of group C, and the Aw at 18 days was about 20%. In contrast, the Aw of group D2 given α-Zn-TCP increased about 15% for 1 day, and then decreased. The Aw at 10 and 15 days was 45% and 8%, respectively. The Aw of group D3 given β-Zn-TCP increased about 20% for 1 day, and then decreased. The Aw at 10 and 15 days was 60% and 37%, respectively. The result indicated that the healing process in the groups given the α-ZnTCP and β-ZnTCP suspensions was shorter than that in the groups administered ZnSO4 or the control.
The time required for the sable to fall from the skin (Toff) was defined as a therapeutic score for the repair of thermal burns. Toff was evaluated based on visible observations and values are shown in Figure 8. The result indicated that the Toff was 14.5 days, the best for the α-ZnTCP suspension, and their order was α-ZnTCP > β-ZnTCP > ZnTCP > nontreatment.
In vitro Zn release tests (Fig. 3), the Zn release from ZnSO4 was very fast, reflecting high solubility of ZnSO4 in water. In contrast, the Zn release from α-ZnTCP and β-ZnTCP were very slow (Fig. 3), respectively. Specific surface areas of β-ZnTCP was about 20 times larger than that of α-ZnTCP, but the Zn release of β-ZnTCP was much slower than that of α-ZnTCP. Because, α-ZnTCP and β-ZnTCP did not directly dissolve in saturated calcium and phosphate solutions, such as a SBF, but those transformed gradually into hydroxyapatite, stable form (the data not shown), and then Zn ion was released slowly from the crystalline solids (Fig. 3), respectively. The Zn release rate of α-ZnTCP was significantly higher than that of β-ZnTCP, because β-ZnTCP transformed into α-ZnTCP at high temperature as mentioned in the experimental section, indicating that the α-ZnTCP had higher chemical potential than the β-ZnTCP. In in vivo tests, ZnSO4 sample solutions were injected to the group D1 (ZnSO4-administrered model) divided 14 times for 2 weeks to avoid too high plasma Zn concentration, but the maximum plasma Zn levels was significantly high at 2.8 μg/mL on day 6, the levels were still high at around 1.7 μg/mL on days 8–15 (Fig. 4, left). In contrast, the group D3 had only initial shots, however, the profile of the plasma Zn levels in the group D3 (the β-TCP administered) was almost the same as that of the group C (no administrated), and much lower than that of group D1. The profile of the group D2 (the α-TCP administered) was between the group D1 (ZnSO4 administrated) and D3 (the β-TCP administered). Because a too high plasma Zn concentration induces serious side effects on cells, the range of Zn concentrations for an optimal biological response, or the therapeutic index, is very narrow, so, there might be a high risk of side effects from ZnSO4 administration. In contrast, the α-TCP and the β-TCP administrations might be controlled their plasma Zn levels depended on their material characteristics. Because the in vitro Zn release profiles of ZnSO4, α-ZnTCP and β-ZnTCP was significantly different (Fig. 3), reflecting their solubility in water, the Zn-AUC of ZnSO4 administration was about two times higher than that for α-ZnTCP and β-ZnTCP (Fig. 4, right).
In contrast, Krötzsch et al.12 reported that the ALP activity was an acute inflammation marker because the enzyme levels were increased in acute wounds, but not in chronic inflammation processes. They concluded that the ALP activity was an important factor to be considered in acute inflammation in wound injury and their healing process in chronic wound repair. So, the ALP of the rats with thermal burns was measured and evaluated as an index of cell activity. The ALP profiles of all rats were fluctuated (Fig. 5, left) because the ALP values were slightly affected by fluctuation of phosphate and calcium levels in plasma, but that of the group D1 rats (ZnSO4) was significantly higher than those of the other groups (D2, D3, and C).
The ALP-AUC of ZnSO4 (Fig. 5, right), was significantly different from those of α-ZnTCP, β-ZnTCP, and C, because cell activity was linked with a suitable plasma Zn level, and a too high plasma level reduced cell activity because a too high plasma Zn concentration can have serious side effects on cells.13 The range of Zn concentrations for an optimal biological response, or the therapeutic index, is very narrow, so, there might be a high risk of side effects from by ZnSO4. Because the maximum Zn plasma concentrations for α-ZnTCP and β-ZnTCP (Fig. 4, left) were much lower than that for ZnSO4 administration reflecting a controlled slow in vitro Zn release from the calcium phosphate devices (Fig. 3), the slow Zn release patterns of α-ZnTCP and β-ZnTCP were significantly effective on therapeutic score, the Aw at local tissue sites (Figs. 7 and 8), and might be accelerated to repair wounded area by thermal burns treatment. The result suggested that ZnSO4 did not affect the cells at the local tissue site because Zn ions distributed rapidly to the whole body, but the α-ZnTCP and β-ZnTCP suspensions might be activated local cells reflecting their slow release patterns. In initial stage at day 5, the difference between the Aw values of optimal Zn released (D2 and D3) and the control groups (C) were not significant, but the former were slightly lower than the latter, so, it may be that the impaired immune functions on the wounded site at initial stage was inhibited by an adequate zinc supplementation from the ZnTCP suspension.
The α-ZnTCP and the β-ZnTCP consisting of a calcium phosphate matrix incorporating Zn ions were meta-stable calcium phosphates under a physiological condition, and exhibited a slow Zn release in SBF from the matrices. This study demonstrated the efficacy of the ZnTCP suspension in shortening the healing of thermal burns when injected in zinc-deficient rats. These materials allow for the possibility of controlling systemic and local effects through the period of administration. The results indicated that the therapeutic effect of the slow release of Zn from calcium phosphate materials was significantly more effective than the injection of Zn solutions. The materials could provide for the better treatment of thermal burns.