In vitro and in vivo tests of PLA/d-HAp nanocomposite

The bioactivity of the PLA/d-HAp nanocomposite with 30 wt.% d-HAp was evaluated by in vitro tests and indicated that after 7 immersion days in SBF solution, PLA amorphous part was hydrolyzed and PLA crystal part was remained. The formation of apatite on the surface of the material was observed. The in vivo test results of PLA/d-HAp nanocomposite (70/30 wt/wt) on femur of dogs displayed that 3 months after grafting, the materials did not induce any osteitis, osteomyelitis or structural abnormalities. The histological and x-ray image demonstrated a growth of the bone into the material area, while osteitis and osteomyelitis were not observed.


Introduction
Over the past decade, hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 , HAp) has been widely used as bioceramic for bone tissue engi neering due to osteoconductivity, biocompatibility, excellent bioactivity, chemical and structural similarity to the mineral phase of native bone [1][2][3][4]. However, its clinical applications have been limited because of brittleness, difficulty of shaping; extremely slow degradation in vivo [5]. Recently, the fabri cation of nanocomposites based on HAp and biodegradation polymers is attracting the attention of scientists because of their ability in replacing the metal and alloy implants [6].
Poly(lactic acid) (PLA) has been chosen for tissue engi neering scaffold due to their good biodegradability and bio compatibility [7][8][9]. The final degradation products of PLA are H 2 O and CO 2 , which are neither toxic nor carcinogenic to the human body and eliminated by natural way. Further PLA has high elastic modulus which is higher than that of natural cancellous bone [10]. In the nanocomposite, PLA plays a role to improve mechanical property and HAp contributes by resembling the natural microstructure analogous to those of bone.
PLA/HAp nanocomposites were fabricated by many methods such as: emulsion method, melt mixing, high pres sure processing, electrospinning and solvent casting method [11][12][13]. In these methods, solvent casting method was used commonly because it does not require expensive equipment, can create a big amount of products.
Advances in Natural Sciences: Nanoscience and Nanotechnology

In vitro and in vivo tests of PLA/d-HAp nanocomposite
In vitro bioactivity of PLA/HAp nanocomposites can be evaluated by immersing the material in saline [14], phosphate buffered saline (PBS) [15] and the simulated body fluids (SBF) [3,[16][17][18]. Validity of the material can be best observed under in vivo conditions after its implantation in an organism. These results showned that after 12 weeks implanted in femur bone of a Wistar rat, the hydrolysis of PLA and the formation of new bone were observed. Simultaneously, the collagen fibers were formed at sites where PLA was hydrolyzed. Thus PLA/ HAp nanocomposites behaved as the natural bone which are phagocytosed and resorbable, they can be considered as bio compatible [7,9].
In this work we used nanocomposite containing PLA, mag nesium and zincdoped hydroxyapatite (dHAp), poly(ethylene oxide) (PEO) and xenetic with the ratio 70/30/5/10 (wt/wt/ wt/wt) to investigate the formation of apatite on the surface of material immersed in the SBF solution and their weight changes were also discussed. The in vivo research was carried out on femur of dogs. The body temperature, femoral radio graphs before and after implantation were investigated.

Experimental
In previous research we investigated and chose a suitable con dition to synthesize PLA/dHAp nanocomposite by solvent casting method [19]. PLA/dHAp nanocomposite (70/30 wt/ wt) has E modulus of about 550 MPa and the tensile strength of 18 MPa.
The material was fabricated for testing in vitro in the SBF solution and in vivo on femur of dogs. In order to observe the materials during the implantation on femur of dogs, the material must have a photoresist capacity. Hence, the photore sist (xenetic 10 wt.%) was added into the nanocomposite. The gel paste mixture containing PLA, dHAp, PEO and xenetic (70/30/5/10 wt/wt/wt/wt) into dichloromethane (DCM) was pelleted (10 × 15 × 0.2 mm 3 ) (PLA/dHAp). The pellets were sterilized before testing in vitro and in vivo.

In vitro test
The in vitro degradation property of PLA as well as the for mation of apatite on the surface of PLA and PLA/dHAp nanocomposites were evaluated in the SBF solution. 1 l of SBF solution was prepared according to typical procedure [3,[20][21][22] by using the following materials: 8 g NaCl; 0.35 g NaHCO 3 ; 0.4 g KCl; 0.48 g Na 2 HPO 4 · 2H 2 O; 0.1 g MgCl 2 · 6H 2 O; 0.18 g CaCl 2 · 2H 2 O; 0.06 g KH 2 PO 4 ; 0.1 g MgSO 4 · 7H 2 O and 1 g glucoza were dissolved in distilled water. The pH of the SBF solution is 7.4 (this value is in the pH range of the human body fluids pH = 7.35-7.45) [23]. The samples of PLA or PLA/dHAp nanocomposites were immersed in the cell containing 40 ml SBF, and kept at 37 °C, during different immersion times: 1, 3, 7, 14, 21 and 28 d. Then they were gently rinsed with distilled water before being dried with 24 h at room temperature. pH of SBF solution, weight loss and SEM images of the samples were determined.
The mass of PLA and PLA/dHAp nanocomposites before and after immersed in SBF solution was determined by Precisa XR 205 SMDR analysis balance. The pH value of SBF solu tion was measured by using pH3110 Meter.
The surface of PLA/dHAp nanocomposites before and after immersion in the SBF solution were examined by using Hitachi S4800 scanning electron microscope (SEM).
The phase component of PLA and PLA/dHAp before and after 7 immersion days in SBF solution were analyzed by xray diffraction (XRD) (Siemens D5000 Diffractometer, CuKα radiation (λ = 1.540 56 Å) with step angle of 0.030°, scanning rate of 0.042 85° s −1 , and 2θ degree in range of 10-60°.     The sensors were connected to the signal amplifiers (Bio Amps) and signals were collected by using the Powerlab (data acquisition system) and Labchart software (AD Instrument, Australia).
Data was analyzed by using Labchart software, temper ature results were average of about 3 min data recording.

2.2.
2. X-ray image recording method. Animals were anes thetized by using ketamine (5 mg kg −1 ), and lying on the table. The left leg was lifted up, pulled to opposite side to avoid overlapping image.
Animal femur xray images were taken by using following parameters: shooting dose of 60 kV, current density J = 35 mA s and distance D = 1 m, film size: 24 × 30 cm 2 , placed just behind the femur.

Electrocardiogram (ECG) recording method.
Animals were anesthetized by using ketamine (dose of 5 mg kg −1 , intramuscular injection). Dog was supine on the operating table, shaving and clean in four dog's soles.  ECG data were analyzed by using module ECG analysis in Labchart software.

Hematological and biochemical indices analysis
method. Hematological indices were analyzed by using Swelab Alpha Hematology analyzers, airline Swelab, Sweden in 2014.

In vitro test of PLA/d-HAp nanocomposites
The in vitro degradation of PLA as well as the formation of apatite on PLA and PLA/dHAp nanocomposite (70/30 wt/wt) into the SBF solution was evaluated by the variation of the pH (figure 1). The obtained results showed that the pH of two solutions decreased according to immersion time. The pH value of the solution containing PLA decreased more than that of the nanocomposite. It can be explained as follows. When these materials were immersed into the SBF solution, two pro cesses occurring simultaneously: the first process expressed by following equations Surface ECG electrodes were attached to dog's leg follow recording rule. The electrodes were connected to the signal amplifiers (Bio Amps) and signals were collected by using the Powerlab (data acquisition system) and Labchart software (AD Instrument, Australia).
is hydrolysis of PLA to generate lactic acid, and release H + ion, the second process is the formation of HAp, which consumes OH − ion. Both of processes reduced pH of the SBF solution.
The pH value of the SBF solution containing PLA was slower than that containing PLA/dHAp nanocomposite because of the presence of PEO as a compatibiliser which made the interaction between PLA and dHAp better and the hydrolysis of PLA in PLA/dHAp nanocomposite became more difficult than neat PLA sample. Besides, dHAp in the component of the nanocomposite was crystal nucleation to promote process of formation of apatite crystals on the sur face of material. The formation of new apatite crystal also hinders interface of PLA with the SBF solution, leading to the slow hydrolysis of PLA in the nanocomposite. Furthermore, the decrease of the pH solution can be explained due to the formation of apatite on the surface of the nanocomposite: the hydrolysis of PLA released H + ion, leading to the dissolution of dHAp in the following equation The concentration of ions forming HAp (Ca 2+ , HPO 2− 4 , OH − ) increased in the surrounding SBF, which promoted the forma tion of apatite crystal.
The variation of weight of PLA and PLA/dHAp nano composite during immersion time was displayed in figure 2. The weight of PLA decreased correspondingly to the nega tive mass change. This result showed that the hydrolysis of PLA was more dominant than the formation of apatite. For the PLA/dHAp (70/30 wt/wt) sample, the hydrolysis process of PLA in 3 immersion days happened strongly, leading to decrease in the weight of the sample. However, after 7 and 10 immersion days, the weight of the sample was higher than that of 3 immersion days, it showed that during this time, the formation of apatite was stronger than the hydrolysis of PLA. The weight of this sample after 14 immersion days was approximately with that of the sample before immersing (Δm = −2 × 10 −5 g). The variation of this sample weight after 21 immersion days had positive value which indicated that the formation of apatite was more dominant than the hydrolysis of PLA. This can be explained by the formation of apatite crystals on the surface of PLA/dHAp which hinders the interface of PLA with the SBF solution. Figures 3 and 4 displayed SEM images of PLA and PLA/ dHAp nanocomposite (70/30 wt/wt) which were immersed in the SBF solution with different immersion times. It is clear that apatite was formed on the sample surface. With the neat PLA sample after 7 d immersed in the SBF solution, the new apatite crystals were observed on the surface of the sample and formed thick block after 21 immersion days. However, SEM images still showed sites where PLA was not covered by apatite crystals (figures 3(b) and (c)).
The SEM image of PLA/dHAp nanocomposite (70/30 wt/ wt) before soaking in the SBF solution indicated that dHAp crystal in the nanocomposite was cylinder shape. After soaking in SBF solution, the formation of the new apatite crystals on the sample surface was flakeslike shape and the surface of PLA/dHAp nanocomposite was nearly full covered by the apatite crystals after 7 immersion days. Specially, after 14 and 21 immersion days in SBF solution, the apatite crystals were full covered and uniform arrangement on the surface of PLA/ dHAp nanocomposite. This result also proved good compat ibility of PLA and HAp in PLA/HAp/PEO nanocomposite prepared by solvent casting method. Figure 5 presented the XRD pattern of PLA and PLA/ dHAp nanocomposite before and after 7 immersion days in   the SBF solution. The XRD pattern of PLA (curve 1 in figure 5) showed that PLA is a semicrystalline polymer. After 7 immer sion days in SBF solution, PLA amorphous part was hydro lyzed and remained PLA crystal part with two characteristic peaks at 2θ = 16.92° and 2θ = 19.50° (curve 2 in figure 5) [12]. The XRD pattern of PLA/dHAp nanocomposite after 7 immersion days (curve 4 in figures 5) indicated the character istic peak of HAp at 2θ = 31.99°. Further, two peaks of PLA crystal at 2θ = 16.53° and 2θ = 18.99° were observed. There was a shift of these peaks in the nanocomposites in the com parison with PLA sample after 7 immersion days in the SBF solution. It can be explained by molecular interaction between dHAp and PLA such as specific hydrogen bonding between -C=O group in PLA with -OH group of dHAp and bonding between COO-group in PLA and Ca 2+ of dHAp. This result is completely agreement with the results of change of mat erials weight aforementioned.       figure 7. It indicated that PLA/dHAp material areas had lower density than the surrounding medulla and outside cortex of bone. Outer bone cortex had highest density, medulla had lower density than cortex and material had lowest density. In xray images, we measured density of the material and compare with that of medulla and cortex of bone. This parameter was used to evalu ate the absorption of PLA/dHAp material in dog's body [24].

3.2.1.4.Hematological parameters.
Three days after surgery, red blood cell (RBC) count and hemoglobin (Hgb) concentra tion in dog decreased slightly in comparison with those before surgery (table 2), but within normal limits [25]. Some causes such as anesthetics and pain from the wound induced less nutrition that leads to red blood cell and hemoglobin decline. This result shows that surgery had a little effect to the dog and this is an usual result in femur surgery. White blood cell (WBC) count in dog increased signifi cantly in comparison with that before surgery. After surgery, blood vessels and bone lesions caused acute inflammatory response of the dog body. The cells responsible for phago cytosis process, such as neutrophil, monocyte, lymphocytes were activated, proliferated and entered the bloodstream to approach damage tissues. On the third postoperative day, platelet count increased in comparison with that before surgery (table 3). This is a result of hemostatic process after surgery, the dog body launches blood coagulation to heal and against hemorrhage. This is also a normal reaction of the dog body after clinical interventions [25,26]. These results showed that there is an acute inflammatory reaction and blood coagulation, but no anemia on the third postoperative day [22]. The results also showed that monocytes increased and lymphocytes declined on the third postoperative day. It indicated that there is an acute inflammatory response against foreign antigens in dog's body. However, this response is moderate, and does not cause serious disturbances to the animals.

Three months after surgery.
3.2.2.1.Hematological parameter. Red blood cells, white blood cells, platelet counts and hemoglobin concentration at 3 months after surgery were similar at 1 and 2 months after surgery. Thus, PLA/dHAp materials existing 3 months in dog's femur did not induce infection or affect to hematopoi etic function (table 5). These results were normal and consis tent with some previous authors [25,27].
Count and percentage of WBC at 3 months after surgery were similar to those at 2 months after surgery (table 6). These data are equal to normal indices in healthy animal without surgery.  PLA/dHAp material existing 3 months in femur did not induce chronic infection that presented by neutrophils, mono cytes, eosinophils and lymphocytes being in normal range. Further, it did not stimulate allergic and toxicity reactions to the body, presented by no change in eosinophils and basophils. These results clearly demonstrate the biological compatibility of materials in the animal body for long periods.

3.2.2.2.ECG parameter in three months after surgery.
ECG results in 3 months after surgery showed that the heart rate about 103 cycles min −1 (tables 7 and 8). Duration and ampl itude of P, T wave, the QRS complex and PQ, QT, ST interval are equal to in 1 and 2 months after surgery and in the nor mal range of ECG in dogs [28]. These results indicate that the PLA/dHAp material did not induce abnormal of heart conduction, depolarization of the atria, ventricles and bundle branch.

3.2.2.3.Biochemical indices of liver and kidney function.
Level of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), urea, creatinine, protein and albumin in serum on third postoperative month were the same at first and second postoperative month (table 9). This data showed that the body has adapted to the presence of material and there is no irritation or interaction that affect to liver and kidney function.

3.2.2.4.Image of dog's thigh area and femur after three months
of surgery. In our experiment after 3 months of surgery, the study subjects were healthy, good scar, without the phenom enon of leakage, drainage, material push out and rough skin at the incision. There was no calcification, necrosis in tissue around grafts region. Exposed through the muscle layer, the periosteum can see white material attached to the bone itself and surrounding bone grew into material area ( figure 9).
There is no hole and osteonecrosis spot in the bone marrow and the periosteum. Surrounding bone tissues were ripples shore and pressed into the material.

3.2.2.5.Microscope image in PLA/d-HAp area after three
months of surgery. After 3 months of surgery, there is a fewer inflammatory cells (neutrophil, macrophage, monocyte, eosinophil, basophil) exist around material area in femur bone.
Periosteum often has reactions such as thickness, rough ness, flaking and shelling in inflammatory area [29,30]. In this study when observed on periosteal microscopic images, we saw smooth periosteum and no periosteal inflamma tory reaction. These results indicated that PLA/dHAp mat erial had good biological compatibility and did not cause the chronic inflammatory of bone [31].
Microscope images also showed bone formation reaction around material area (figure 10). There were many osteo blasts, collagen fibers and thickwall blood vessels around material area. This result is similar to the process that occurs on bone healing normally, without complications [32].
3.2.2.6.Animal weight after three months of surgery. After 3 months of surgery, under nurtured conditions of animal center, Military Medical University, animal weight was significantly higher than before surgery and 1st month. This result showed that animals were not affected by the surgery and materials in the femur (table 10).

3.2.2.7.X-ray dog's femur image after three months of surgery.
In xray image, there is no abnormal morphological in dog's femur at 1st, 2rd and 3rd month after surgery. This result indi cated PLA/dHAp material had high biological compatibility   (1) 10.54 ± 0.67 One month after surgery (2) 10.92 ± 0.59 Two months after surgery (3) 12.36 ± 0.71 Three months after surgery (4) 14.17 ± 0.52 ( p 1-2 > 0.05, p 1-3, 1-4, 2-4 < 0.05). in both phase acute and chronic. It did not induce stimulation, inflammation, rejection reactions on the dog bone site. In xray image, density of PLA/dHAp material was lowest, bone cortex is highest, medulla was medium. However, over 3 months of surgery the density of PLA/dHAp material became higher, the difference between the materials and the medulla became increasingly lower. These results demonstrate PLA/ dHAp materials in femur were absorbed increasing gradually over time ( figure 11).

Conclusion
The variation pH of SBF solution, material mass, morphology, phase structure of PLA/dHAp nanocomposites in SBF solu tion showed the formation of the HAp on the surface of the nanocomposites and the hydrolysis process of PLA after immersion in SBF solution. The in vivo test results of this nanocomposite on femur of dogs during 3 months proved PLA/dHAp nanocomposites with good biocompabitility and promising potential applications for bone implant.