EFFECT OF CISSUS QUADRANGULARIS ON FRACTURE HEALING : AN ANIMAL STUDY

Dr. Rahul Mahajan, Dr. Sanjeev Gupta, Dr. Nitin Choudhary, Dr. Neeraj Mahajan, Dr. Vishal R Tandon, Dr. Jyotsna Suri and Dr. Ghanshyam Dev 1 Senior Resident,Deptt of Orthopaedics,Govt Medical College, Jammu. 2 Prof and Head,Deptt of Orthopaedics,Govt Medical College(GMC) Jammu. 3 Resident, Deptt of Orthopaedics,Govt Medical College(GMC) Jammu. 4 Lecturer, Deptt of Orthopaedics,Govt Medical College(GMC) Jammu. 5 Prof, Deptt. of Pharmacology,GMC Jammu. 6 Prof, Deptt of Pathology,GMC Jammu. 7 Ex-HOD, Deptt of Radiodiagnosis & Imaging, GMC Jammu. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 April 2020 Final Accepted: 07 May 2020 Published: June 2020 Traditionally Cissusquadrangularis has been used as a medicinal plant since antiquity. Cissus has been used in various Ayurvedic classical medicines to heal broken bones and injured ligaments and tendons. It is being used very commonly & without much clinical evidences of level1. The said drug is being widely used by orthopaedic surgeons.The current study was done on 30 healthy male albino rabbits after clearance from IAEC.The animals were divided into two groups viz. control and study group. Fibula of left leg of all the animals were fractured surgically. The study groups received oral CissusQuadrangularis in solution. Fracture healing of study group was compared with the control group radiologically and histopathologically to establish the role of CissusQuadrangularis on fracture healing.

Traditionally Cissusquadrangularis has been used as a medicinal plant since antiquity. Cissus has been used in various Ayurvedic classical medicines to heal broken bones and injured ligaments and tendons. It is being used very commonly & without much clinical evidences of level-1. The said drug is being widely used by orthopaedic surgeons.The current study was done on 30 healthy male albino rabbits after clearance from IAEC.The animals were divided into two groups viz. control and study group. Fibula of left leg of all the animals were fractured surgically. The study groups received oral CissusQuadrangularis in solution. Fracture healing of study group was compared with the control group radiologically and histopathologically to establish the role of CissusQuadrangularis on fracture healing.

…………………………………………………………………………………………………….... Introduction:-
The biology of fracture healing is a complex biological process that follows specific regenerative patterns and involves changes in the expression of several thousand genes. In animal models (rat, rabbit, mouse) the peak of soft callus formation occurs 7-9 days post trauma with a peak in both type II pro-collagen and proteoglycan core protein extracellular markers (2). At the same time, an intramembranous ossification response occurs subperiostally directly adjacent to the distal and proximal ends of the fracture, generating a hard callusthat ultimately provides the fracture with a semi-rigid structure which allows weight bearing (3).

Cissus Qudrangularis
It is a perennial plant of grape family. It is also known as veldt grape, devil's backbone, adamant creeper, asthisamharaka, hadjod and pirandai.
It is probably native to India, Sri Lanka and Bangladesh, but is also found in Africa, Arabia and Southeast Asia. It has been imported to Brazil and the southern United States.

Materials And Methods:-
Present study was conducted in department of Orthopaedics Government Medical College Jammu during the period from July 2016 to November 2017 on 30 albino rabbits weighing 1-2 kgs, to study the effect of cissusquadrangularis on fracture healing. 1: Animals: In the present study, 30 healthy male albinorabbits of 1-2 kg weight and around 2 yrs of age were taken as experimental animals. (FIG 1) The rabbits were procured from the central animal house of the department of pharmacology, GMC, Jammu.
All the CPCSEA guidelines were followed. All the animals were kept in standard cages (FIG 2) and maintained under standard laboratory conditions (temperature 25+/-2°c with 12 hrs light/12 hrs dark cycle) with free access to standard diet and water throughout the study. 2: Drugs: CissusQuadrangularis was procured from the market & 10mg/kg drugwas given tostudy group as dissolved solution.

Experimental design:
Thirty rabbits, taken as experimental animals, were divided by randomization into two groups of fifteenrabbits each and each group was as follows: Group I(n=15) :-served as control and received normal saline. Group II(n=15) :-served as study group and received10 mg/kg of cissusquadrangularis orally once daily for 4 weeks.

Methodology:-
Healthy male albino rabbits between 1-2 kgs were taken for study. The animals were divided in two groups (I & II) as follows:-Group I = Control 15 animals Group II= Study 15 animals The animals were kept in iron cages in a room (Fig. 2). They were fed with standard diet. The body weight of rabbits were recorded before the onset of experiment. The animals of Group I were not givencissusquadrangularis. Group II were given 10mg/kg solution of cissusquadrangularis orally.

Preparation Of Drugsolution:
Drug solution was prepared by dissolving cissusquadrangularis in normal saline before oral administration.

Administration of thedrug:
CissusQuadrangularis solution was administered to the rabbits with a syringe/cannula.

Anaesthesia, surgical fracture of fibula of rabbit and itsdissection:
After anaesthesia with Inj.Ketamine 35mg/kg i/m and Inj. Xylazine 5mg/kg i/m. Under all aseptic precautions part was prepared (Fig. 3,4,5 &6). A small longitudinal skin incision was given over lateral aspect of middle of leg (Fig.7&8).Soft tissue dissected.Fibula exposed (Fig. 9).A transverse fracture of middle 1/3 of fibula done with help of bone cutter (Fig. 10).Skin closed with 3-0 ethilon (Fig. 11). ASDsdone (Fig. 12), Analgesics(Inj. Meloxicam 0.2mg/kg i/m B.D.) + antibiotics (Inj. Ampicillin 25mg/kg i/m B.D.) were given for one week post surgery.Radiographs were taken. Oral cissusquadrangularis administered to the study group daily for 4 weeks.Regular ASDs done. Radiographs of all animals were taken on day 0. (Fig. 13&14) 370 Radiographs of 8 animals fromeachgroup were taken at 2weeks (Fig. 15&16)& the segment of fibula of these animals were dissected for histological assessment (Fig. 19,20,21&22). The remaining animals (7 in each group) were still under study and the animals from study group were administered with the drug under study for two more weeks.
Theremaining animals' radiographs were taken at 4weeks (Fig. 17&18) and the segment of their fibula dissected for histological assessment. (Fig 23&24).
Measurement of frature healing: Parameters:

1.
Radiological:Radiographs taken serially at 0,2 & 4 weeks to see the callus formation and fracture union.

2.
Histological: By using modified Allen's grading system. Results:-Changes in each group observed & compared on radiographs & histological examination( By Allen's fracture healing scoring system).
Radiographic comparison of two groups: (Table 1) No grossdifferencewas seen on plain radiographs between both the groups at 2 weeks . Both the groups showed similar callus formation. (Fig. 15&16).
However at 4 weeks radiographs of 3 out of 7 samples of study group showed complete union and 4 out of 7 showed incomplete union whereas all the samples of control group showed incomplete union. (Fig. 17&18). Histological comparison of two groups:( Table 2 & Table 3  The data is shown in mean+/-SEM, Mann-Whitney U Test was applied and p<0.05 was considered significant;p<0.01 was considered highly significant(HS).So results of the present study are highly significant statistically at 2 weeks in favor of the said drug. Table 3:-Comparison of Mean (SD) in Allen's Fracture Healing Scoring system at 4weeks between two Groups: The data is shown in mean+/-SEM, Mann-Whitney U Test was applied and p<0.05 was considered significant; p<0.01 was considered highly significant. As per Mann-Whitney U, p at 4 weeks is 0.01 which is also highly significant as p<0.05. So results of present study are also highly significant at 4 weeks in favor of the drug under study. *SD=Standard Deviation, SEM=Standard Error of Mean.  The results at 2 weeks are significant clinically as well as statistically. The results at 4 weeks are significant clinically but not significant statistically due to small sample size as per Fisher's Exact Test.

Discussion:-
CissusQuadrangularis has been used as a medicinal plant since antiquity. It has been used in various Ayurvedic classical medicines to heal broken bones and injured ligaments and tendons. In siddha medicine it is considered a tonic and analgesic, and is believed to help heal broken bones. Deka D K et al(1994) in their while evaluating the effect of methanol extract of cissusquadrangularis on experimentally fracture radius and ulna of dog revealed that cissusquadrangularis treated animals record faster initiation on healing process than the control animals both on radiological and histological examination. The results are in accordance with current study.

Summary And Conclusion:-
1. On radiographic comparison between study and control groups, there was not any gross difference in callus formation at 2 weeks. However, at 4 weeks 3 animals out of 7 in the study group showed complete union of the fracture unlike controls. The results were significantly better in favor of animals treated with the test drug with p value <0.05. 2. The mean Allen's Fracture Healing Score in both the groups in 2 weeks treated animals and 4 weeks treated animals showed highly significant scores in comparison to the control grouprespectively with p value <0.01 thereby indicating a better and early callus formation histo-pathologically in favor of cissusquadrangularis treated animals in comparison of controls. 3. On grading the histological Allen's fracture healing score , better signs of histological fracture healing were seen in study group with 50% animals from the study group showed >1 score at 2 weeks and all the animals showed Score (Allen's) Control Study 0 or 1 8 4 >1 0 4