THE IMPACT OF HYDROXYAPATITE SINTERING TEMPERATURES ON THE STRUCTURAL AND FUNCTIONAL CAPACITY OF EMBRYONAL LUNG FIBROBLASTS

Abstract

The specific aim of this investigation was to investigate the effects of hydroxyapatite (HA) sintered at different temperatures on the proliferation, morphology, and cellular alterations of Embryonal Lung Fibroblasts (MRC-5) cells in culture. Microcrystals of HA were prepared by following standard laboratory protocols. The calcined HA was then placed on an automatic Tyler sieve stack to select for particles between 1-40 μm in size. The calcined material was sintered at 700, 1000, 1200 and 1300 °c for 24 hours. The sintered material (1.0 grams of HA) was added to a total of 20 mg of L-lysine as a binder. Finally, this combination was cold-pressed into cylindrical form using a 3/8 inch die set at a compression load of 5000 kg to form a disk of HA. Calculated density of the devices was directly proportional to temperature of sintering. MRC-5 fibroblasts were obtained from the American Type Culture Collection (ATCC). Proliferation Assessment, morphological evaluations were conducted following standard lab protocols. Cells were analyzed for Maliondialdehyde (MDA) levels by utilizing thiobarbituric acid reactive substance (TBARS) measurements. Results of this study revealed that there was an initial (at 24, and 48 hours) increase in proliferation rate observed in wells containing HA sintered at 700, 1000, and 1300°C. There were no significant differences in proliferating cells among control and capsules sintered at 1200°C (P<0.05). However, at 72 hours there was 2-3 fold increase in cell number for the cells encountering 1200°C compared with control and experimental wells. Cellular membrane damage was evident in cells exposed to HA sintered at 700, 1000 and 1300°C, but not for control or HA 1200°C treated cells. Morphologically, the cells showed evidence of progressive fragmentations, cellular debris and lysis at 72 hours for cells in the HA sintered groups of 700, 1000 and 1300°C. HA sintered at 1200°C did not cause changes in cell morphology for the duration of the experiment. In conclusion, the results of this investigation suggest that sintering temperature is essential factor in the development of a high mechanical strength HA delivery systems. The results from this observation and our previous findings using different calcium phosphate devices suggest that the use of optimal HA density is crucial for material constructs to replace or repair tissue defects.

INTRODUCTION

In recent years, the use of Bioceramics in dental and orthopaedic fields have received remarkable attention [1-7], This can be attributed to the ceramic materials favourable properties to an in vivo environment. These includes corrosion resistance, ease of fabrication [3-9], and hardness, to name afew. Furthermore, the growing trend toward using the ceramic materials as implantable devices may just be the shape of things to come. Unlike other implantable materials, several studies have shown that ceramic particles produce less cell reaction than extensively used polymer or metal particles. Previous studies conducted in our laboratories [1-18] and others have suggested that in designing compatible ceramic devices, it is essential to devote special attention toward providing a proper response to ceramic-implant interface. Ideal implantable ceramic-based devices must be highly acceptable by the host in order to ensure highest and prolonged functional activity of surrounding tissues. The hypothesis of this investigation elutes that porosity and mechanical strength of hydroxyapatite (HA) ceramic drug delivery devices is highly impacted by the temperature of sintering during the fabrication process. The goals of this study are: (i) to develop an effective HA ceramic drug delivery system to release a variety of biologicals at a sustained level for long duration and (ii) to alleviate side effects associated with conventional means of administering medication. The specific aims of this investigation was to explore the effects of HA sintered at different temperatures that might have on the proliferation, morphology, and cellular alterations of Embryonal Lung Fibroblasts (MRC-5). MRC-5 cell line was used extensively as a model to assess the biocompatibility of implantable devices [1-7]. MRC-5 is a diploid human cell culture line composed of fibroblasts derived from lung tissue of a 14-week-old aborted Caucasian male fetus. The MRC-5 cells themselves are known to reach senescence in around 45 population doublings. MRC-5 cells are also used to produce several vaccines including Varicella and Polio. Infected MRC-5 cells secrete the virus, and can be cultured in large volumes suitable and an excellent model for various medical conditions.

MATERIALS AND METHODS

Fabrication of Ceramics:

Microcrystals of HA were prepared by following standard laboratory protocols [1,2]. The calcined HA was then placed on an automatic Tyler sieve stack to select for particles between 1-40 μm in size. The calcined material was sintered for 24 hours using high performance programmable furnace. The sintered material (1.0 grams of HA) was added to a total of 20 mg of L-lysine. Finally, this combination was cold-pressed into cylindrical form using a 3/8 inch die set at a compression load of 5000 kg to form a disk of HA. The final density of devices ranged between 1.680 (700 °C) −2.076 (1300 °C) gm/cm³.

Cell Line:

MRC-5 fibroblasts were obtained from the American Type Culture Collection (ATCC). The principles of sterile techniques and preparation of culture media, steps in trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, and counting cells using a hemacytometer, were performed using our lab protocols unless otherwise indicated.

Experimental Design:

The design is illustrated in table 1. Proliferation assessment, total MDA levels were conducted following methods of Benghuzzi et al [1-5], A total of 150 MRC-5 (50,000 cells/well) loaded wells were divided into 5 groups containing 10 wells each. Group one served as sham control (no HA), while groups 2-5 were loaded with HA devices sintered at various temperatures as in indicated in Table 1.

Table 1:

Experimental Design

Phase I	Cell number	Time (n=10 wells)/phase
Control (no HA)	50,000	24, 48, 72 hours
700 °C HA	50,000	24, 48, 72 hours
1000 °C HA	50,000	24, 48, 72 hours
1200 °C HA	50,000	24, 48, 72 hours
1300 °C HA	50,000	24, 48, 72 hours

Morphological Evaluation:

Histopathological evaluations were performed on control and experimental groups at three phases (24, 48, and 72 hours). At the end of each phase, the cells were harvested with the removal of the supernatant and set on ice. The remaining cells in the wells were rinsed with 1 ml of phosphate buffered saline (PBS) and scraped to ensure removal of cells from the sides and bottom of the wells. These cells were collected into a labeled microfuge tubes. The MRC-5 cells rinsed with an additional 0.5 ml of PBS and added to the corresponding labeled microfuge tubes. The MRC-5 cells in the labeled microfuge tubes were then centrifuged for 10 min. at 1000 RPM. After removal from the centrifuge, the pellets were resuspended in 400 μl of PBS. Four labeled slides were prepared for each tube, 3 wells/phase (n = 12 slides per group). A total of 100 μl of cell suspension were placed upon each slide and heat fixed. A routine of H&E was employed to assess the morphologic changes associated with various HA devices exposure.

Maliondialdehyde (MPA):

MDA measurement is an essential marker for lipid peroxidation. Samples were analyzed for malionaldehyde bis diethyl acetal levels through the utilization of thiobarbituric acid reactive substance (TBARS) measurements [4,8,11], One hundred microliters of sample or standard were added to 1 mL of 20% TCA and allowed to set for 2 minutes. This was followed by the addition of 1 ml of 0.67% thiobarbituric acid in 0.5N NaOH. The mixture was vortexed and heated to 100 °C for 30 minutes. After the incubation period, the samples were cooled and centrifuged. The absorbance was recorded at 532 nm and compared to the standard curve. Concentrations were determined and were reported as μM TBARS.

Statistics and Graphics:

Statistics analysis was performed using Sigma Stat Software. Graphics were produced using Slide Write Plus Software.

RESULTS AND DISCUSSION

The results of this study revealed significant findings regarding the role of sintering temperature of HA on the viability of MRC-5 cells. Figure 1 demonstrates that there was an initial (at 24, and 48 hours) increase in proliferation rate observed in wells containing HA sintered at 700, 1000, and 1300°C. There were no significant differences in proliferating cells among control and devices sintered at 1200°C. However, at 72 hours there was 2-3 fold increase in cell number for the cells encountering 1200°C compared with control and other experimental wells. This observation and previous observations using different calcium phosphate carriers [1,10-17] suggest that sintering HA at 1200 °C can be considered an optimal temperature in fabricating the devices. Further evidence was observed in the viability status of the cells during the three periods. Figure 2 indicates that cellular membrane damage was evident in cells incubated with HA sintered at 700, 1000 and 1300°C, but not for control or HA 1200°C exposed cells. Morphologically, the cells showed evidence of progressive fragmentations, cellular debris and lysis at 72 hours for cells in the HA sintered groups of 700, 1000 and 1300°C. Cells exposed to HA sintered at 1300 C showed evidence of fewer small, round shaped cells with hyperchromatic nuclei, signs of remarkable cell injury (hydropic swelling), prominent nucleoli, fragmented cytoplasm and cytoplasmic extensions. HA sintered at 1200°C did not cause changes in cell morphology for the duration of the experiment. MRC-5 cells in the exposed HA sintered at 1200 °C exhibited distinct healthy characteristics including small size, predominantly round shape, and hyperchromatic nuclei with evidence of few elongated cells in a debris background. Table 2 shows a comprehensive screening of H&E stained cells in all groups (representative images illustrated in Figure 3).

Figure 1:

The Effects of various HA sintering-temp on the proliferation rate of MRC-5 cells in culture. It was obvious that at 72 hours: cell counts for all groups were similar to control except 1200 °C which remained several fold higher.

Figure 2:

The Effects of Various HA Sintering-Temp on the MDA levels of MRC-5 cells in culture. Under chronic conditions the cells incubated with HA sintered at 1200°C had the greatest proliferation and least damage when compared to the other groups.

Table 2:

Effects of Various HA Sintering-Temp on the Morphology of MRC-5. Evaluations was conducted by screening 10 views per slide. Histopathologically, the screening reveals an optimal temperature of sintering at 1200 °C. For more details, see methods section.

Figure 3:

Representative images demonstrating the effects of various HA sintering-temp on the morphology of MRC-5. Morphologic and analysis showed the cells incubated with the 1300°C HA devices to have undergone lysis or other extreme cellular alterations (see table2).

CONCLUSIONS

Sintering temperature and cellular density are essential factors in the development of a high mechanical strength HA delivery systems. The results from this observation and our previous findings using different calcium phosphate devices suggest that the use of optimal cell density is crucial for material constructs to replace or repair tissue defects.