J Bagh College Dentistry Vol. 32(2), June 2020 Histomorphometric 26 Histomorphometric evaluation of the effects of local application of red cloveroil (trifolium pratense) on bone healing in rats Nada M.H. AL-Ghaban (1), Ghazwan H. Jasem (2) Article DOI: https://doi.org/10.26477/jbcd.v32i2.2891 ABSTRACT Background: Red clover oil (Trifolium pratense) has isoflavones bunches which have estrogen-like exercises and may establish an option in contrast to hormone substitution treatment. The present study investigated the impact of Red clover oil on bone healing in rats by histomorphometric study. Materials and Methods: Intra bony defect was performed in right femur of thirty six healthy male albino rats. Then these rats were randomly divided into three groups (12 rats each): one control and 2 experimental groups. For control, the bony defect was left for normal healing; for the experimental (S) group, the defect was treated with hemostatic absorbable gelatin sponge; and for experimental (RS) group, the bony defect was treated with 0.2 ml red clover oil and covered by haemostatic absorbable gelatin sponge. Six rats from each group were sacrificed at 2 and 4 weeks intervals. Histomorphometric analysis was performed on H&E bone section of all the studied groups which includes counting of bone cells (osteoblasts, osteocytes and osteoclasts), trabecular number, trabecular area and bone marrow space area. Results: Histomorphometric results of bone cells revealed that the combination group stimulated larger numbers of osteoblasts and osteocytes than in sponge and control group. Number of new bone trabeculae, trabecular area and bone marrow space area showed higher mean values in combination groups than others. Highly significant differences between groups were observed in all histomorphometric parameters throughout all durations. Conclusion: Red clover oil stimulated larger numbers of osteoblasts and osteoclasts, indicating increased bone remodeling especially at 2 weeks interval as compared with sponge and control groups. Key words: Bone defect, Red clover, Rats. (Received:15/8/2019; Accepted:24/9/2019) INTRODUCTION Bone mending is a profoundly effective procedure that takes into account the scar less recovery and redesigning of imperfections identified with the treatment of injury, pathology, or inborn variations from the norm. Bone fix was a multistep procedure including relocation, expansion, separation, and initiation of a few cell types.(1) Bone redesigning requires the relations between numerous bone cells to revamp, protect, or direct bone quality or potentially mineral homeostasis in the light of adjusting natural impacts. There were four discrete stages to this procedure: initiation, retention, inversion, and development with ingestion; that occurs through osteoclasts and osteoblasts, correspondingly.(2) Bone imperfections attempt accommodating recuperating strategy through synchronized accompanying advancement of skeletal and vascular segments in a delicate cartilage callus setting. Among this setting, (1) Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. (2) Master Student, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. Corresponding email, nada_gaban@yahoo.com bone restoration restates a few of the equivalent cell and sub-atomic systems that delivers the embryonic bone.(3) Red clover (Trifolium falsification) is a perpetual herb developing in all mild and subtropical zones in the world over. In a few societies, it is utilized as customary prescription, other than its daidzein and genistein content, red clover shows a high substance of methylized forerunners: biochanin A and formononetin.(4,5) Isoflavonoids mixes present in red clover oil are the primary dynamic substances of "phytoestrogens". Epidemiological and clinical research revealed constructive outcomes of isoflavone utilization over bone with the danger of building up a few osteoporosis.(6) This study aims to examine the impact of Red clover oil on bone healing in rats histomorphometrically. MATERIAL AND METHODS: Thirty six males healthy albino rats, aged between (4-5) months with weight ranged between (250-300 mg), were used in this experimental study. All rats were kept under supervision and nursing from the staff of the animal house of Biotechnology Research https://doi.org/10.26477/jbcd.v32i2.2891 J Bagh College Dentistry Vol. 32(2), June 2020 Histomorphometric 27 Center/University of Al-Nahrain, Baghdad, Iraq. All experimental procedures were conducted in accordance with the ethical approval of animal experiments of College of Dentistry, University of Baghdad. Intra bony defect of about 2 mm in width and 3 mm in depth was performed in right femur of each rat.(7) Then rats were randomly divided into three groups (12 rats each): 1) Control group (C): the bonydefect was left for spontaneous normal healing. 2) Experimental group(S): the bony defect was treated with hemostatic absorbable gelatin sponge. 3) Experimental group (RS): the bony defect was treated with 0.2 ml(8) red clover oil (Trifolium pratense) and covered by haemostatic absorbable gelatin sponge. Then six rats from each group were sacrificed at the end of recommended periods (2 and 4 weeks). The right femur was dissected and the soft tissue was removed to expose the entire bone to be cut at 5 mm away of the defect sides. The bone specimens immediately were stored in 10% freshly prepared formalin and left for 2 days for fixation. Bone decalcification was performed by using formic acid sodium citrate solution, which was prepared freshly from 2 solutions (125 cc formic acid 90%, 125 cc distilled water and 50 mg sodium citrate, 250 cc distilled water).(9,10) Then, bone tissue was dehydrated with alcohol and embedded in paraffin. Sections of 5 μm were prepared in the usual fashion, and stained with hematoxylin and eosin. Histological examination was performed using light microscope. Histomorphometric assessment of bone cells (osteoblast, osteocyte and osteoclast), trabecular area, trabecular numbers and bone marrow space area was performed by software program (Image J. exe), which is an image processing program developed at the National Institutes of Health.(11) RESULTS Histological results A- Control group: Histological view of bone defect revealed sparse of bone trabecular coalesce with cutting bone in control group of 2 weeks duration. Osteocytes in newly formed trabecular enclosing areas of marrow tissue and large number of osteocytes are embedded in bone (fig.1). At 4 weeks duration, the osteoblasts were noticed at peripheries of the thick bone trabecular and osteocytes inside these bone (fig.2). B- Sponge group (S): The histological examination of this group after 2 weeks duration illustrated the deposition of bone trabeculae that replaces areas of bone defect (fig.3). At 4 weeks, the osteoblasts are seen at peripheries of the bone, osteocytes seen arranged in circular around Haversian canal (fig.4). C- Combined red clover oil and sponge group (RS): Histological examination of this group after 2 weeks revealed thick well developed bone trabeculae filled defect area with numerous blood vessels and inflammatory cells inside bone marrow (fig.5). At 4 weeks mature bone filled defect area by presence of osteon (fig.6). Statistical analysis of histomorphometric findings Tables (1 and 2) show comparison differences using ANOVA and LSD test among all studied groups at different healing periods for bone parameters trabecular area (BTA), trabecular number (TN) and bone marrow area (BMA) in control(C), sponge (S) and combination (RS) groups in both healing periods (2 and 4 weeks). The result showed a highly significant difference between all groups in both durations (two and four weeks) for these three bone parameters. J Bagh College Dentistry Vol. 32(2), June 2020 Histomorphometric 28 Figure (1): Control group at 2-weeks showing basal bone (BB), blood vessels (BV), inflammatory cells (IC) and osteocytes (OC). H&EX40 Figure (3): Sponge group at 2-weeks showing new bone trabeculae (BT), osteoclasts (OCL) and inflammatory cells (IC). H&E X40. Figure (5): RS group at 2-weeks showing new bone filled by osteocytes (OC) lined by osteoblasts (OB), blood vessels (BV), inflammatory cells (IC), osteoclasts (OCL) and reversal line (RL). H&EX40 Figure (2): Control group at 4-weeks showing osteoblasts (OB) lined Haversian canal and osteocytes (OC) filled bone. H&E X40 Figure (4): Sponge group at 4-weeks showing regular arrangement of osteocytes (OC) around Haveresian canal (HC) and osteoblasts (OB). H&EX40 Figure (6 ): RS group at 4-weeks showing mature bone (osteon formation) by regularly arranged osteocytes (OC) around Haversian canal (HC) and Osteoblasts (OB) seen riming haversian canal (HC). H&EX40 J Bagh College Dentistry Vol. 32(2), June 2020 Histomorphometric 29 Table 1: ANOVA group comparisons for bone parameters. According to the ANOVA test, there was a highly significant difference among all studied groups in both 2 and 4 weeks intervals in bone cells except for osteoclasts at 4 weeks duration which was significant as shown in table (3). DISSCUSION Within the last decade the use of natural supplements has become more widespread in the search for viable alternatives to existing treatments. Red clover (Trifolium pratense) is a medicinal herb containing flavonoids and isoflavones. Red clover contains at least 9 isoflavones including formononetin, biochanin a (glycosides), daidzein and genistein (aglycones) which promote the formation of Variable Duration Group comparisons F -test p- value BTA 2 weeks 33.5 0.0003 4 weeks 42.9 0.0005 TA 2 weeks 29.4 0.0002 4 weeks 19.7 0.0001 BMA 2 weeks 52.3 0.0006 4 weeks 112.7 0.00008 Variable s Dura -tion Gro ups Comparisons M.D P- value Sig. Osteobla st 2 week C/ S -10.4 0.003 HS C/ RS -11.8 0.0091 HS S / RS - 11.03 0.0075 HS 4 week C/ S -6.8 0.0013 HS C / RS -7.0 0.0011 HS S / RS -6.92 0.004 HS Osteocla sts 2 week C/ S -0.10 0.006 HS C / RS - 0.12 0.0002 HS S / RS -0.11 0.0016 HS 4 week C/ S -0.24 0.0023 HS C /RS -0.29 0.02 S S / RS -0.26 0.0064 HS Osteocyt es 2 week C/ S 3.8 0.0019 HS C /RS 4.4 0.0055 HS S / RS 4.1 0.0028 HS 4 week C/S -9.7 0.0017 HS C/ RS -10.4 0.0059 HS S / RS -10.1 0.0094 HS Vari ables Duratio n Groups Comparisons M.D. p- value Sig. BTA 2 weeks C/ S -3.8 0.0002 HS C/ RS -3.0 0.001 HS S / RS -4.4 0.0004 HS 4 weeks C/ S -9.5 0.001 HS C/ RS -7.4 0.0001 HS S / RS -4.9 0.003 HS TA 2 weeks C/ S -5.6 0.006 HS C/ RS -8.2 0.0011 HS S / RS 3.2 0.0041 HS 4 weeks C / S 1.7 0.0001 HS C / RS 2.2 0.0021 HS S / RS 2.0 0.0087 HS BMA 2 weeks C /S 0.737 0.0043 HS C/ RS 0.654 0.0062 HS S / RS 0.587 0.0028 HS 4 weeks C / S 0.231 0.007 HS C / RS 0.311 0.0028 HS S / RS 0.259 0.0091 HS Table 3: LSD group differences in bone cells. Table.2: LSD Group Differences in Bone Parameters J Bagh College Dentistry Vol. 32(2), June 2020 Histomorphometric 30 bone. (12) Cellular processes stimulated include chemotaxis, mesenchymal cell proliferation and differentiation, angiogenesis, and synthesis of extracellular matrix, although different isoflavones compounds are closely related structurally and functionally. (13) Portrayal of the recovered bone tissue is regularly performed by histological assessment with light microscopy, following standard recoloring of the example. Enlightening histology is utilized to give a general appraisal of the tissue of enthusiasm, giving information with respect to cell morphology, structure and course of action inside the interface with the extracellular grid or with an embedded material.(14) In red clover oil and sponge group (GS) treated defect area showed more and thicker bone trabeculae than that of other groups. It has been reported that isoflavones compounds could increase the osteogenic effect by increasing the osteoblast cell proliferation and stimulating matrix activity. (15) A previous study (16) has been performed to evaluate the performance of isoflavones compounds as a scaffold in bone regeneration procedures to be a promoter of osteoblastic formation and is readily resorbed by osteoclasts. Direct bone matrix anchorage has been shown with collagen fibers deposited in the micropores. Resorption of isoflavones compounds has been reported in a rabbit model with cells having a characteristic of osteoclast cells activity. Histomorphometry permits quantitative examination of histological information, to be specific with respect to length and separation, territory and number of the segments of intrigue. (17) Mean values of trabecular area and number recorded in this study were higher in combination with ( RS ) group, more clearly observed in 4 weeks duration which may seem to be in line with histomorphometric results of Ochiuto et al., 2007 (18) who stated that the increase in osteogenesis seen during the transition from the 14 to 30 days of observation and the total areas of the newly created bone trabeculae showed that the groups submitted to bone filling biomaterial (osteoconduction and autogenous bone graft) showed bone trabeculate area values higher than the control group of the same animal. The present study revealed that the number of osteoblasts was highest in combination (RS) group when compared to others especially in 2 weeks interval. The number of osteocytes increases with time in all studied groups especially in combination group at 4 weeks interval. These results could be explained by the direct action of red clover oil on the differentiation and maturation of osteoblasts and accelerating rate of matrix deposition and its corresponding calcification. These findings agree with Bharathi & Baby, 2017 (19); they found that isoflavones compounds could increase the rate of bone ossification. It is thought to affect bone metabolism by promoting the proliferation of osteoblasts and the synthesis of osteon, which leads to the inhibition of the differentiation of osteoclast- like cells. CONCLUSION Red clover oil is osteoinductive herbal material that promotes and accelerates bone healing process by an early bone formation and maturation. Histmorphometric parameters for all groups showed highly significant difference in overall indicators of bone micro architectures which include trabecular area, trabecular number, bone marrow space area, osteoblasts, and osteocytes numbers. Conflict of interest: None. REFERENCES: 1. 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Phytochem.2017;6(4):79-83. الخالصة : إصالح األنسجة العظمية هي عمليه معقده متعددة الخطوات تشمل التضاعف , الهجرة الخلوية ,التفعيل والتعضية للعديد هدفال الجزء المصاب مجموعه االيزوفالفين الموجودة في زيت البرسيم األحمر يقودها باتجاه التمعدن وأعاده خلق . من انواع الخاليا تساعد في زيادة المكون الملحي للعظم ,القوه الميكانيكية , وزن العظم والكثافة العظمية بواسطة رفع مستوى مصل الفوسفات .القاعدية - 300( شهور ووزن بين4-5لسليمة بعمر يتراوح بين )تم استخدام ستة وثالثين من ذكور الجرذان البيضاء ا :المواد والطرائق تم اجراء االختبار المناعي النسيجي إلظهار الببتيد المؤيد للكوالجين من النوع . ( ( ملغموالتي استخدمت في هذه الدراسة250 . االول على عينات عظميه لجميع المجموعات المدروسة والتي شوهدت خالل اسبوعين من تقدم تم دراستها التي العظم في الصفيحات العظميةاشارت النتائج النسيجية الى ترسب :النتائج التمعدن مع مرور الوقت وتم تحديده بشكل اكبر في مجموعه زيت البرسيم االحمر المغطى باإلسفنجة الماصة من مجموعه د كبير من الخاليا العظمية في مجموعه زيت كشفت النتائج النسيجية للخاليا العظمية عن وجود عد. االسفنج ومجموعه التحكم البرسيم االحمر المغطى باإلسفنجة الماصة (والتي نتجت من إعداد اكبر من الخاليا العظمية المولدة لتكوين العظم مقارنه نوع االول اظهارا إيجابيا كما أظهرت النتائج الكيمائية المناعية لنوع الببتيد المؤيد للكوالجين من ال. بالمجموعتين االسفنج والتحكم أعلى في مجموعه زيت البرسيم االحمر المغطى ايجابيا وقد أظهرت تعبيرا.متزايدا في خاليا أنسجه نخاع العظم و الخاليا العظمية . باإلسفنجة الماصة مقارنه بمجموعتين االسفنج و التحكم خصوصا في فتره األسبوعين من ضاستخدام الموضعي لزيت البرسيم األحمر يسرع في تكوين ونضج العظم ويزيد اي كشفت الدراسة أن اال االستنتاجات: . االظهار االيجابي للببتيد المؤيد للكوالجين من النوع االول في الخلل العظمي أكثر من العملية الفسيولوجية الطبيعية