Effect of Cold Plasma on Histological Compositions of the Rabbits Fracture Bone Tissue

In this research, the rabbits' bones have been fractured, the rabbits were exposed to the cold plasma for five minute times two weeks. The microwave plasma voltage which was used in the search was "175v" and the gas flow was" 2 L" at the room temperature. A Biologic parameters such as alkaline phosphates (ALP), osteocalcin, vitamin D (Vit. D) and calcium (Ca), Triglycerides (TG), Cholesterol (T.chol.), Estradiol and Glucose have studied in its serum. Physiological parameters were measured to prove the effects of plasma on the fracture bone tissue and show the amount of damage and the effect of plasma therapy before and after exposure to plasma.


Introduction
The direct application of plasma in medicine is to understand the physical, chemical and biological mechanisms of direct non-thermal plasma interaction with living tissue. Engineering research has resulted in many advances in health care. Ionizing radiation and laser are examples of a technological breakthrough that have created a diagnosis and new treatments for the disease. Non-thermal plasma medical techniques also have important therapeutic effects and lead to new medical diagnostic tools. Some of these factors include deactivating pathogens, stopping bleeding without damaging healthy tissue, promoting wound healing and treating cancer [1,2,3]. The fracture on the long bone of the
Iraqi Journal of Science, 2019, Vol. 60, No. 9, pp: 1997-2002 1998 rabbits is treated with plasma to improve effect of cold plasma on healing the bone. The microwave plasma is the plasma with "high frequency electromagnetic radiation in the GHz range". Mazhir et al. showed that there was no side effect on the tested animals after exposure to microwave plasma [4]. Plasma is a hot ionized gas consisting of approximately equal numbers of positively charged ions and negatively charged electrons [5]. Microwave plasma or non-thermal plasma is used here. Microwave plasma is a type of plasma that has high frequency electromagnetic radiation in the GHz range. Microwave generated plasma system chooses over other kinds of plasma sources because they are electrodeless plasma, so the task of replacing or cleaning the electrodes and filaments is averted [6]. Microwave discharges produce non-equilibrium plasma since the electrons can respond to the oscillations of the electric field whereas the ions are not able to respond due to their large mass. So most of the microwave energy goes preferentially to the electrons, and then produce plasma far from thermodynamic equilibrium [7]. The aim of this research is to study the effect of plasma on the fracture of rabbits' bone tissues

Methodlogy And Material
The microwave plasma generated from this system was applied on rabbits which shows in Figure  Fracture Bone: It is broken or cracked bone because of the strength exerted against the bone stronger than the bone resistance, which destroys the structure and strength of bone which leads to loss the function of the main bone and leads to pain, sometimes bleeding and the most common sites for fractures of bones are the wrist, ankle, and hip [8]. Histological Composition of the Bone Tissue Calcium: Calcium is one of the most important minerals in the body and is responsible for, bone stiffness, immune defense [9]. It is a basic base for building strong bones and teeth and gives shape and maintenance to bones and teeth and helps in muscle contraction and causes osteoporosis in adults, short-term calcium deficiency can lead to osteoporosis in the bones where the bones deteriorate and there is an increase in fractures.Lack of calcium in the blood (low blood calcium) can be caused by kidney disease or vitamin D deficiency [10].
Absorbance of sample Absorbance of Standard Vitamin D: Vitamin D works like a hormone it helps to make calcium available for bone growth. It promotes intestinal absorption of phosphorus and calcium, vitamin fat soluble the excess is stored in the liver and fatty tissues can be synthesized by the body by exposure to UV light from the sun, the main sources being yogurt, yolks, liver and fish [11]. Therefore, vitamin D is necessary because its deficiency causes rickets and osteoporosis [11,12].

Alkaline Phosphatase (ALP):
Alkaline phosphatase is an enzyme found in all tissues of the body but is mostly concentrated in the bones, liver, kidneys, placenta, and intestines. It is implicated in bone calcification and has an important role in the growth of teeth and bones because it is main for mineralization, mineralization occurs when minerals, such as calcium and phosphorus are deposited into bones and teeth to help them become hard bone . Alkaline phosphatase is a useful parameter for monitoring changes in bone synthesis, the normal level of alkaline phosphatase in the blood is 20 to 140 units / liter, this can alter from laboratory to laboratory [13]. The studies indicate estrogens and androgens act via different cellular mechanisms, the bone-sparing effect of estrogen is antiresorption by inhibition of osteoclast action [14]. Osteocalcin: Osteocalcin is a bone protein (bone matrix) produced by bone cells during bone formation during the late stages of bone formation, bones are constantly remodeled through processes close to bone restoration and formation to maintain the skeleton, dynamic balance disorders such as osteoporosis can lead to bone loss and fractures, bone metabolism can be monitored by measuring the biochemical indicators of bone turnover in the blood, marks provide dynamic and fast procedures for skeletal condition or fracture risk assessment. The osteocalcin measurements are used to assess the rate of bone formation in clinical investigations [15]. Blood Glucose: The relationship between diabetes (high blood glucose levels) and osteoporosis in all clinical and experimental conditions has been documented with a decrease in diabetes in cell proliferation and collagen synthesis during the early stages of fracture healing causing these unknown effects, but insulin has a major role to play in healed fractures through insulin receptors in bone cells and was able to enhance bone stiffness [16].

.(5) Result and Discussion
Table-1 shows the statistical values for the alkaline phosphates, Triglycerides, Cholesterol and glucose for Fracture group. Figure-2 shows the statistical level value for the fracture group for the alkaline phosphates, Triglycerides, Cholesterol and glucose.  The results are shown in Table-1 for fracture group indicate that there is significant change (p value <0.05) in the ALP of control group as compared with infected group ''rabbits with fractured bones''.
There is significant change in ALP for infected group compared with treated group. There are nonsignificant changes (p value >0.05) in Tchol., TG and glucose that means the cold plasma did not effect on these biological parameters Tchol.,TG and glucose this is clearly shown in Figure-   The results in a Table-2 show that there is significant change (p<0.05) in the Ca of the control group as compared with infected group. There are significant changes in Ca of infected group compared with treated group. There is non-significant change (p>0.05) in osteocalcin of control group as compared with infected and treated group.This can be shown in Figure Figure-3 shows the level of vitamin D and estradiol of the fracture group. The result from the Table-3 shows that there is non-significant change (p>0.05) in the vit. D and estradiol in the control group as compared with infected group and treated group. This can be illustrated in Figure-

Conclusion
The results showed that the fracture rabbits bones infected with the cold plasma. The physiological parameters demonstrated a response to the treatment. The ALP had a significant changing and showed a response to the plasma. The result clarified that TG, T.chol, and Glucose had no response to plasma since the p-value was greater than 0.005. The value of osteocalcin illustrated no response to the plasma in the fractured bone since the p-value was greater than 0.005; while the Ca response to the plasma, Vit. D and Estradiol have no significant change.