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In vitro levels of calcium, phosphate and alkaline phosphate activity in media of rat osteoblasts grown in the presence of various implant materials.

Introduction

Artificial bone and prostheses are used for the rehabilitation and restoration of function in the human body. The materials used for implants and artificial bone are evolving. However, a 'one-type-fits-all" material for implants is still eluding scientists worldwide. Materials used have been made from stainless steel [1], cobalt-chrome alloys (Co-Cr) [2], and titanium alloy (Ti), cp Titanium [3], inter alia.

Most of the implants used today are generally successful. With some patients, however, revision surgery or removal of implants after several years may be needed. This is especially probable where fixtures are subjected to considerable torque and movement. Hence, reports of implant success, with any material, must be interpreted with caution. Most implants exhibit immediate integration. Note, for example, that short term success was recently found when Co-Cr and Ti implants were fixed in pigs [4]. However, the host response, over a period of time and under different environmental conditions, affects overall outcome. This may be so because of the nature of chronic rejection, a poorly understood chronic inflammatory and immune response against implants in general. The adaptive immune system confronts a combination of slowly released antigens that are dispersed at very low concentrations. Antibodies and cell mediated immunity responses are designated by many researchers as the number one cause of implant failure [5,6,7].

Several materials are being tried in the search for a better product. For example, tantalum [8], ceramics [9], and composite materials [10] are some of those studied for osseointegration and applicability. Artificial bone made from wood has also been investigated. Several studies have addressed the ability of wood- natural or treated-to withstand the rigors of orthopedic or dental implantation. Investigations with pinus wood [11], beach wood [12], birch [13], and Juniper [14],inter alia, have led to information about wood's good (comparable to bone) mechanical performance (insertion torque and pullout force) [11], fracture behaviour (Young's modulus and failure load under compression) [12], and its stability/ decomposition behaviour--the latter being indirectly represented by wood's favorable osteoconductive [13] and osseointegration [14-16] properties, necessary for bone fusion.

The idea of wood being used to replace bone is appealing since wood is an inexpensive natural product. Additionally, the physical structure of wood is porous and somewhat similar to bone. The hierarchical microfibre structure of cellulose, hemi-cellulose, and lignin has variable porosity and high strength even at low densities [15]. Researchers in Italy have devised a novel system for converting wood to a scaffold of hydroxyapatite [16]. Their method basically involves slow pyrolysis at 1000[degrees]C to reduce the material to a carbon frame, followed by conversion of this frame to hydroxyapatite. In another study, wood was pretreated differently [13]. The organic components were removed by heating in a much lower temperature range, specifically between 200[degrees]C and 140[degrees]C. Under these conditions, there was more integration and less fibrosis found at the interface of the bone and implant when applied in rabbits. At this temperature, the reactive interface of wood would be composed mainly of hydroxyl groups, unlike the carbon frame expected at 1000[degrees]C.

The use of polymethylmethacrylate (PMMA) as a bone cement has persisted since 1959 [17]. PMMA in preformed beads containing benzoyl peroxide is mixed with methyl methacrylate (MMA) containing N, N-dimethyl-p-toluidine (DMPT).

Alkaline phosphate activity (ALP) measurements have been used as a marker for bone formation. It is produced by osteoblasts to provide a high P[O.sub.4.sup.2-] concentration at the cell surface during mineralization. Osteoblast maturation is associated with the expression of ALP [18]. The presence of [Ca.sup.2+] and an increase in inorganic phosphate have been found in the extracellular vesicles at the stage of precipitation of hydroxyapatite or mineralization [19].

The aim of this study was to determine if there is a difference in the production of ALP, [Ca.sup.2+], and P[O.sub.4.sup.2-] for several different materials, over various time periods. The data was analyzed in two-factor ANOVA tests using SPSS V17. The significance of the main and interaction effects is assessed at the ~ =0.05 critical level.

Materials and Methods

All the materials used in this research were procured from the standard companies. This research proposal was approved by the ethics committee, The University of the West Indies, Faculty of Medical Sciences, Trinidad (Ref number: EC65:21/12-06/07).

Cell cultures of rat osteoblasts were grown in the presence of discs (0.8 x 0.25 mm) made of wood and of the various implant materials currently used. The control experiment had no disc. In this, the cells were cultured in the presence of the growth medium alone. Scanning Electron Microscope (SEM) images and surface elemental analyses were performed on all disc materials, before they were placed in and after they were removed from the cell cultures.

Cell Harvest

Calvaria from eight newly born Sprague-Dawley rats were cut out from the skull under a dissecting microscope. The suture areas were removed in such a way as to obtain only the central component of the bone. This method has been described previously [20]. The periosteum and endosteum of the dissected bone were gently scrapped off and washed with sterile [Ca.sup.2+]-free and [Mg.sup.2+]-free Tyrode's solution (Sigma-Aldrich). The bone fragments were cut into fragments of 0.5 mm before being subjected to enzyme digestion. This latter involved the treatment with 2.5 mL of 1.0 mg/mL trypsin (Sigma-Aldrich) in Phosphate Buffer solution (PBS) for 10.0 minutes. The action of the trypsin was stopped by the addition of 5 mL of Medium 199, with glutamine and Earle's salts (MEM), containing 10% fetal calf serum (FCS) (all from Sigma-Aldrich). The liquid was decanted and 5.0 mL of PBS added. The bone fragments were then sequentially digested 5 times using 5.0 mL of 0.25% collagenase (Fisher Bioreagents) in PBS for 20 minutes with slight agitation in a water bath at 37[degrees]C. In each instance the bone fragments were allowed to settle after 3 minutes. The last three digestions was pooled, then centrifuged at 200g for five minutes to allow for pellet formation. This pellet was washed using 5.0 mL of PBS before being resuspended in 10.0 ml of growth medium. This consisted of MEM containing 10% FCS and 0.2% antibiotics (100 units penicillin, 100 [micro]g/mLstreptomycin, and 0.25 [micro]g/ mL amphotericin B) (Sigma-Aldrich).

Experimental Template

One milliliter of the osteoblast cell suspension in the growth medium was evenly distributed in each well of a 24-well sterile cell culture plate, containing discs of Ti (American Elements, Merelex Corp.), Co-Cr alloy (Nobilium Inc.), PMMA (SuperDental Co.) and wood (Pinus). Five (5) replicates of each disc (and of the blank medium) were used. All the discs used for the experiments were made to identical dimensions: 0.8 x 0.25 mm. The wood discs were pre-treated by placing them in an oven for 12 hours at 200[degrees]C. All the wells were topped up by the addition of 2.0 mL of growth medium. This experimental template was placed in a humidified incubator at 37[degrees]C under an atmosphere of 5% C[O.sub.2] and 95 % air. Aliquots (1.0 mL) of the medium were taken from each well at 1, 3, 6 and 7 days and replaced with 1.0 mL of fresh growth medium. Samples collected were frozen at -20[degrees]C for analysis. The activity of ALP and the concentrations of [Ca.sup.2+] and of P[O.sub.4.sup.2-] were obtained using Ortho Vitros dry slides Chemistry Analyzer (VITROS[R] 4600).

Electron Scanning Microscope Analysis

The composition and images of the surfaces of all discs were obtained before they were placed in the experimental template. After seven (7) days, each disc was removed from the experimental template with osteoblast cells and its image and surface composition recorded. All discs were vacuum dried then coated with 15-40 nm gold (Denton Vacuum Desk II). The surface compositions and images were recorded using a Scanning Electron Microscope (Phillips SEM 515) set at 30 KV. The data collected was analyzed using Genesis (EDAX) software.

Results and Discussion

The results indicate that ALP was produced by the osteoblast cultures grown in the presence of all of these materials. Cells were observed covering all test materials after five days.

The mean levels of the markers, [Ca.sup.2+], P[O.sub.4.sup.2-], and ALP activity, after various times (1, 3, 5, 7 days) are given for the different disc materials in Figure 1.

Electron Microscope Analysis

The primary cell cultures adhered to the base of the wells and to the surfaces of all discs. This occurred within two hours after the cells were introduced into the wells. Confluence was also seen in all wells after five days of culture. Chemical analysis showed the presence of [Ca.sup.2+] after seven (7) days in all cell compartments. SEM surface analysis revealed (1) osteoblast cells growing on the surface of all disc materials, (2) no [Ca.sup.2+] on the surface of any disc material, including wood, before it was placed in the cell cultures, and (3) the presence of [Ca.sup.2+] on the surface of the wood disc but not on that of any of the Co-Cr, Ti and PMMA discs- Figure 2.

Impacts of 'Disc Material' and 'Time' on Levels of [Ca.sup.2+], P[O.sub.4.sup.2+] and ALP Activity

Figure 1 illustrates these impacts graphically. The results of the two-factor ANOVA tests are summarized in Table I. These test (1) whether there are significant differences between the disc materials in terms of the amounts of [Ca.sup.2+], P[O.sub.4.sup.2-], and ALP activity found in their cell culture compartments, (2) whether Time has an impact on the amounts of these same 3 markers, and (3) whether there is a Time-Material interaction in that the pattern observed over Time is different for the various disc materials. Tests which are significant are designated as 'S' and those that are non-significant as 'NS'. The 'p' values are given in parentheses and matched next to the critical value [alpha] =0.05.

The impacts of the disc material on the levels of [Ca.sup.2+] and P[O.sub.4.sup.2-] were not significant (noreal differences between the levels of [Ca.sup.2+] and of P[O.sub.4.sup.2-] found in cells with discs of different material). With ALP activity, on the other hand, there was a significant impact of the disc material on the levels found in the cell culture medium after 7 days, with the highest activity found in cells with the wood disc (Figure 1).

Culture time impacted significantly on the levels of all markers. This is expected. This research aimed not at establishing the obvious but at looking to see how differences between discs in the levels of all the markers ([Ca.sup.2+], P[O.sub.4.sup.2-] and ALP) changed with Time. This comes from the Time-Material interaction effect. There is a significant interaction effect between the factors of 'Time' and 'Disc Material' for the levels observed in the cell cultures for each of the 3 markers (Table 1). The implication here is that the pattern of each marker's relative values with time changes from one disc material to the next. Hence, the impact of 'Time' on the levels of any marker is different depending on which disc Material is used in the cell culture.

Correlations among Dependent Variables at Different Culture Times

The data for each sampling day was subjected to correlation analysis to see how the relationships among the 3 outcomes- [Ca.sup.2+], P[O.sub.4.sup.2-], and ALP activity- developed over time. The correlation coefficients for pairs of outcome variables are given in Table 2. These were estimated for each day's data using information from all of the 5 individual replicate cell cultures (instead of the mean values) for all 4 disc materials plus the control.

As the culture time increased there appeared to be an increasingly stronger correlation between the level of ALP activity in the cell and the concentrations of either P[O.sub.4.sup.2-] or [Ca.sup.2+], with the strongest correlations in each case (0.465 and 0.807 respectively) obtained for the Day 7 data. The relationship between [Ca.sup.2+] and P[O.sub.4.sup.2+] was also more pronounced (r = 0.771)for the longer culture times.

The experimental method employed in this research followed a standard cell culture technique. The use of cultured osteoblast cells in vitro for experimental research has been carried out for over 40 years. Osteoblast cell lines, for example MG 63, Saos-2, U-2, have been used in many studies involving implants [21]. When these cells were compared to primary cultures of animal osteoblasts, osteoscarcoma cell lines showed distinct differences in behavior [22]. Even when matched against human mesenchymal stromal cells, MG-63 and Saos-2 show difference in ALP activity when grown in the presence of Titanium [23].Experimental templates using primary osteoblast cultures reflect more phenotypic properties of normal osteoblasts than osteoblastic cell lines [24]. However, to acquire osteoblasts for primary cell culture experiments, tissue is usually obtained from prenatal and newly born animals. Osteoblasts can be isolated by migration [25], by mechanical techniques [26], by cloning [27], and by enzymatic digestion, as was employed here. One main concern is that when collagenase is used for over one hour, damage to antigen receptors on the cell membrane occurs. To reduce changes or damage to the cell membrane of the osteoblast cells, the collagenase digestion in this experiment was performed in a sequential manner, at twenty minute intervals. The main advantage in using enzyme digestion in this study was to harvests a large amount of cells in a short time frame. It has also been reported that the population of cells collected is adulterated by fibroblasts [28]. Other studies reported that, in addition to this contamination, there is the problem that osteoblasts isolated by enzymatic digestion fail to show any mineralization in vitro [29,30]. These reports cannot be denied, yet different results have been reported by researchers using similar models [18]. It has been demonstrated clearly that bone cell purity is not a requirement to obtain bone formation in vitro [31]. Although generalized osteogenesis is evident in fetal rats, a differential growth pattern is usually observed at nineteen days in utero. Cells isolated from calvaria are at different stages of differentiation. The cellular composition of the whole calvaria in rats shows that one fifth is composed of osteoblasts. The frontal and parietal bones, otherwise known as the skull cap, are used. There are several disadvantages of primary culture systems. These include low cell yield, time-consuming steps, nonhomogenous population and changes to the cell surface receptors. In this study cell suspension from the last three digest were collected, discarding the first two collagenase fractions. It was thought that by doing this a more homogenous osteoblast population was obtained.

Conclusion

The results show that [Ca.sup.2+], P[O.sub.4.sup.2-], and ALP activity are produced in cells with discs of all materials tested, even wood. The statistical analysis shows further that there is no significant impact on the amount of [Ca.sup.2+] and P[O.sub.4.sup.2-] produced by changing the disc material. The type of disc material does affect significantly the amount of ALP activity within the cell, with the wood disc showing the highest ALP activity after 7 days(Figure 1). Time caused significant increases in the amount of all markers for all disc materials. The rate at which each marker was produced, however, changed from one disc material to the next (the Time-Disc Material interactions were significant for all markers). These results are informative because they show that wood stacks up well against the known and approved materials, such as Ti, Co-Cr and PMMA.

After the seventh day, spectral analysis revealed the presence of [Ca.sup.2+] on wood only. None of the other materials gave similar findings. In contrast to the implant materials that are used today, wood is completely natural. The channel structure of wood creates a porous scaffold that could possibly allow favorable bone growth. Heat treated wood has reactive hydroxyl chemical groups at its interface with bone. These reactive hydroxyl groups on the cellulose chains are available for covalent and for hydrogen bonding. At the level of the bone there are reactive [Ca.sup.2+], P[O.sub.4.sup.2-], proteins and collagen available for interaction with the implant at the ionic level [32]. These results suggest that wood should be explored further as an option to the more standard implant materials in use today.

Acknowledgment

This research was funded by the University of the West Indies Campus Research Fund, grant number CRP.4FEB07.6.

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Fiayaz A. Shaama (1), Shivananda B. Nayak (2), Valerie A. Stoute (3)

(1) Restorative Dentistry, Prosthetics Unit, (2) Department of Preclinical Sciences, Biochemistry Unit, Faculty of Medical Sciences, University of the West Indies, EWMSC, Trinidad and Tobago, West Indies

(3) Departments of Environmental and Graduate Studies, University of Trinidad and Tobago, O'Meara Campus, O'Meara Industrial Estate, Lots 74-98, West Indies

* Corresponding author, Fiayaz Aleem Shaama, fiayaz.shaama@sta.uwi.edu

Received 14 February 2013; Accepted 16 June 2013; Available online 21 July 2013

Table 1: Results of Two-Factor (Disc Material and Time)
ANOVA with replicates

Outcome Measured    Effect          Significance (p value)

                    Disc Material   NS (p = 0.25 > 0.05)
[Ca.sup.2+]         Time            S (p = 0.000 << 0.05)
                    Interaction     S (p =0.012 < 0.05)
                    Disc Material   NS (p =0.44 > 0.05)
P[O.sub.4.sup.2+]   Time            S (p = 0.000 < 0.05)
                    Interaction     S (p = 0.0002 < 0.05)
                    Disc Material   S(p = 0.0000 << 0.05)
ALP                 Time            S(p = 0.0000 << 0.05)
                    Interaction     S(p = 0.0000 << 0.05)

Table 2: Pearson Product Moment Correlation Coefficients for Pairs of
Markers at Different Culture Times

Variable 1          Variable 2          Pearson    Product Moment
                                        Day 1      Day 3

[Ca.sup.2+]         P[O.sub.4.sup.2-]   0.253      0.648
[Ca.sup.2-]         ALP                 0.007      0.229
P[O.sub.4.sup.2-]   ALP                 -0.127     -0.042

Variable 1          Correlation    Coefficients
                    Day 6          Day 7

[Ca.sup.2+]         0.771          0.685
[Ca.sup.2-]         0.391          0.807
P[O.sub.4.sup.2-]   0.352          0.465
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Title Annotation:Original Article
Author:Shaama, Fiayaz A.; Nayak, Shivananda B.; Stoute, Valerie A.
Publication:Trends in Biomaterials and Artificial Organs
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Date:Jul 1, 2013
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