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Incorporation of Lucrin-TPO into Resin Based Composites

The use of resin based composites (RBCs) continues to increase at different rates internationally, partly due to the demise of amalgam. They provide a versatile and robust restoration material [1] [2], however, in recent years the biocompatibility of dental composite restorations has come into question [3]. Degree of conversion and degree of crosslinking are key concepts within RBC formulation as they directly impact the biocompatibility of dimethacrylate resins [3] – the reason for which being the potential leaching effect of individual components from RBC formulations, e.g. monomers, free radicals and photoinitiators [4]. Evidence has shown that improvement of mechanical properties and degree of conversion of conventional photoinitiator systems, i.e. camphorquinone/amine systems (CQ) can be seen with an increase in photoinitiator concentration. However, such increase in concentration is limited by a threshold, after which no further advantages are seen. Alongside this, such an increase often warrants unwanted side-effects i.e. a yellowing effect [5] and potential CQ toxicity [6]. As a result, CQ is now looked at being replaced by alternative photoinitiators such as Lucrin-TPO (TPO) due to their apparent improved degree of conversion (DOC) and degree of crosslinking (DCR) [7] without the need for a large increase in photoinitiator concentration whilst subsequently holding the potential to reduce the issue of component leaching from RBCs. The aim of this current study is to investigate the effects of photoinitiator chemistry on the mechanical properties of RBCs.
Composite preparation
A total of four RBC formulations were used within this project. Of the four RBC’s two different photoinitiators were used: CQ and TPO. RBC 1 and RBC 3 were based on CQ whilst RBC 2 and RBC 4 were based on TPO. Within each photoinitiator group, alternative filler quantities were used. Table 1 provides the detailed compositions of RBCs 1-4.
Curing of composite specimens
Each sample was cured using the light engine (Lumencor, inc®) at an irradiance of 1000 Mw/cm2. The power output to attain such irradiance was calculated using a calibration curve which was obtained using a fibre based spectrometer (USB 4000, Ocean Optics, Dunedin, USA). CQ based RBCs were cured using cyan light at a power output of 70. TPO based RBCs were cured using blue light at a power output of 29. As such, the absorption spectrums of both TPO and CQ could be attained.
Degree of conversion (DC)
DC was measured using static samples via attenuated total reflectance (ATR). The DC for each RBC was calculated three times, with each sample having the following dimensions: 6mm in diameter and 2mm in thickness.
Vickers hardness testing
A silicone cylindrical hardness testing was carried out using the Vickers Hardness testing method (Duramin tester, Struers). Three samples for each RBC formulation (1-4) and two different irradiance times (9 and 20 seconds) were tested after up to 48 hours on both upper (cured) and lower (non-cured) surfaces. Each sample side was tested three times and an average acquired for each irradiance group. Every sample was tested using a load of 1.961N for a period of 10 seconds.
Depth of cure (DoC)
A silicone cylindrical mould with dimensions of: 6mm in diameter and 12mm in depth were filled with experimental composite. Three samples for each RBC and two different irradiance times (9 and 20 seconds) were obtained and then extracted from the mould. The sample was then tested using the ISO4049 method. Such method involves a scrape test whereby all ‘non-cured’ material is scraped off, and the length of the remainder of the sample is measured using a digital calliper. The obtained value was then divided by two.
Statistical analysis
For each data population the standard deviation was calculated. Alongside this, when two sets of data samples were compared against each other, a one-way analysis of variance (ANOVA) was calculated.
Photoactive resins which are cured using a light source utilise photoinitiator systems which absorb light of a certain wavelength, creating excited states which initiate the process of polymerization. It is known that several factors affect the efficacy of RBC curing, e.g. light source and irradiance time; which can be optimized to improve the mechanical properties of dental composites [8].
The incorporation of alternative photoinitiator systems such as TPO into RBC formulation has been proposed to produce a more optimal light cure, and thereby improving the overall mechanical properties of the RBC when compared to the more traditional photoinitiator system of CQ [7]. According to the kinetic parameters of the present investigation, TPO has been shown to produce a higher DC (see Figure 1 and 2) and a higher DoC (see Figure 3 and 4) when compared to CQ. Alongside this, TPO has shown to produce higher hardness values (Figures 5 and 6).
The mannerism by which CQ and TPO absorb light occurs via two different pathways. CQ reacts with its co-initiator (DMAEMA) in its excited triplet state to generate one active free radical, whereas TPO cleaves directly onto the molecule in question, generating two free radicals (6). It is for this reason that despite the same irradiance time, due to the presence of a larger number of free radicals, TPO is shown to generate a higher DC compared to CQ (Figure 1 and 2). Due to the nature of the obtained DC values (via static samples), the rate of polymerization was not attained. However, looking at the difference in DC for TPO between the 1, 9 and 20 second values, the difference is fairly constant compared to those presented by CQ. This would indicate that TPO produces a more constant rate of polymerization in comparison to CQ.
In reference to the ISO 4049 method which was used to assess the DoC for RBCs 1-4, it is assumed that the leftover hardened specimen is not ideally cured, and hence this is accounted for by the division factor of 2 [9]. It can be clearly seen (Figure 5 and 6) that TPO is shown to produce better DoCs in comparison to CQ. This could be indicative of the issue of opacity; CQ is known to have a yellow tinge in colour [10], and thus altering the opacity of the composite compared to TPO. Opacity is a factor which affects light transmission through the material, and such difference in colour could result in CQ producing the visibly lower DoC.
The assessment of RBC hardness post-curing is a common mechanical test used to study the curing efficacy. The obtained results are related to DC [11] and for this reason, it is not surprising that in this study, TPO based RBCs generated higher hardness values compared to CQ based resins (Figure 3 and 4). Alongside this, light transmission through RBCs is affected by surface reflection, absorption and scattering as a result of filler particles, thus a reason for the variation of hardness between both the upper and lower surfaces of the specimens.
The incorporation of TPO into RBCs has shown to be a promising alternative to CQ, producing higher degrees of conversion, hardness and depth of cure. It is important to understand the importance of the different pathways for free-radical formation in dimethacrylate systems. When considering the clinical applications of TPO it is imperative to consider the wavelength outputs of light curing units, which would require a shift towards the UV range.

Diosgenin in the Treatment of Osteoporosis

Diosgenin prevents bone loss on retinoic acid induced osteoporosis rats
Diosgenin has preventive and therapeutic effect on osteoporosis.
Diosgenin can prevent bone loss.
Model group of osteoporosis are successfully induced by retinoic acid.
Object: To observe the preventive and therapeutic effects of diosgenin on retinoic acid induced osteoporosis in rats.
Methods: The rats induced by retinoic acid were used as rat model of osteoporosis. The femoral dry weight, bone calcium (Ca), phosphor (P) contents of femoral were measured; the biochemical index in serum of alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), estradiol and osteocalcin were determined after the rats were given diosgenin at the dose of 10, 30 and 90mg kg-1, respectively, and were compared among the model group, normal group and positive control group.
Results: The osteoporosis rat model was successful induced by retinoic acid. Compared with the model group, the lessening of femoral weight, the short femoral transverse diameter and the bone mineral of diosgenin groups were improved in the diosgenin-treated group. The estradiol and osteocalcin levels in the middle and high dose groups were significantly higher than that of the model group, while the ALP and TRAP levels were lower than the model group.
Conclusions: Diosgenin can prevent the loss of bone in experimental rats. The mechanism may be that it improves the level of estrogenic hormone of estradiol and inhibits the high bone turnover.
Osteoporosis is a bone disease that leads to an increased risk of fracture with reduced bone mineral density (BMD), deteriorated bone microarchitecture, and altered amounts and types of proteins in bone [1, 2]. The purpose of treatment of osteoporosis is to prevent the bone fractures by decreasing bone loss or, preferably, by enhancing bone density and strength [3, 4]. Early detection and treatment of osteoporosis can sufficiently decrease the risk of future bone diseases, while it was difficult to cure the osteoporosis by rebuilding the bone. There was none of an available treatment to cure osteoporosis completely. Therefore, early prevention of osteoporosis is as important as treatment [5].
The model of retinoic acid induced osteoporosis in rats is used in several studies to evaluate the influence of substances on bone loss in human, for its easy operation, high successful rate, short time consumption and type symptoms of osteoporosis [6-9]. Early studies observed that large dose of vitamin A was toxic to the skeletal system of rats [10, 11]. Further studies also showed that retinoic acid causes constant decrease of BMD in a short period of 1-3 weeks [12]. All these findings demonstrated short-term effects of retinoic acid could act as an appropriate revulsive of osteoroposis [13].
Diosgenin, asteroid sapogenin (Fig. 1), extracting from Dioscorea wild yam tubers, such as the Kokoro [14, 15], has been shown to inhibit proliferation, suppress inflammation, and induce apoptosis in tumor cells [16-18]. The aglycone (sugar-free) and diosgenin is used for the commercial synthesis of steroid products, such as pregnenolone, cortisone, progesterone, etc.Previous studies showed that diosgenin could be used to prevent and treat osteoporosis [19, 20]. All these studies indicate the safety and efficacy of diosgenin using as a certain alternative treatment modality for osteoporosis, and it is available for diosgenin being used therapeutically for postmenopausal women who attempt to reduce osteoporotic progression. However, the molecular mechanism of diosgenin activity in bone-derived cells, remains largely unknown.

Fig.1 Chemical structure of the steroid diosgenin
In the present study, we investigated the influence and mechanisms of diosgenin activity in preventing and treating osteoporosis rat model induced by retinoic acid. Our study also provides further information to the possible therapeutic use of diosgenin on the treatment of bone-related diseases.
Materials and Methods
Rats (National Grade A experimental animal) aged 90 days old weighting between 190-260 g were offered by the Center of Experimental Animals, China Pharmaceutical University. All of the animal care and animal experimentations were provided in accordance with the Guide for the Care and Use of Laboratory Animals (National Research and Council, 1996).
Diosgenin was provided by the National Institute for the Control of Pharmaceutical and Biological Products. Retinoic acid, alkaline phosphatase (ALP) reagent kit, tartrate-resistant acid phosphatase (TRAP) reagent kit, and inorganic calcium (Ca) and phosphorus (P) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Osteocalcin labeled by 125I were obtained from Aladdin reagent Co. Primary and secondary anti-estradiol and osteocalcin were purchased from Sigma Co. (Shanghai, China).
Animal model
A total of 40 female rats were treated with the retinoic acid suspension (70 mg/kg) once daily for 14 days. The rat osteoporosis model induced by retinoic acid was examined by the cortex, size and beam of bone.
These rats were randomly allocated to one of four reagents for another 14 days, with 0.5% CMC-Na 70 mg/kg (as the model control), or three doses of diosgenin (10, 30 and 90 mg/kg) as the low, middle and high dose-treated groups, respectively. Additionally, another 10 healthy rats were supplemented as the healthy control with 0.5% 70 mg/kg CMC-Na once daily for 28 days. The dosing was adjusted according to the daily weight conditions. All rats were raised under consistent conditions during the study.
Bone histomorphology
Bilateral femur bones were got from the sacrificed rats for histomorphology analysis. The right one was weighed to analysis the weight index (g/100 g body weight), and the left was cut at the mid-diaphysis to test bone density using dual energy X-ray bone densitometer. Both the diameter and the length of the femur bone were measured.
Bone mineral detection
For mineral detection of bone in different treatment groups of rats, the femur bone was first dehydrated and then carbonized by burning into ashes at 800 ºC for 8 hours. The levels of Ca and P (mmol/g) were determined by inorganic calcium and phosphorus assay kits when some ashes were dissolved in 6N HCl.
Biochemical indexes of serum
The serum samples were obtained from the rats after the last dose of the study drugs given for 24 hours. The levels of ALP and TRAP with their reagent kits were measured, respectively. The absorbance of serum samples was measured by ALP and TRAP kits at 400-415 nm under the base and acid conditions, respectively, to detect their enzymatic activity. The levels of estradiol and osteocalcin were measured by radial immunoassay.
Statistical Analysis
Statistical comparison analysis was performed by students’ t-test. Results were expressed as mean ± SE with significance defined as P < 0.05.
Effect on bone histomorphology
The cortex of bone in normal control group was much thicker than osteoporosis model group treated by retinoic acid. The size of bone was tiny and the beam of bone was thin in the model group (Fig. 2 A