Preparation Of Berberine Chitosan Thermosensitive Hydrogel And Its Therapeutic Effect On Infectious Bone Defects In Canines

Infectious bone defect（IBD）refers to a bone disease caused by factors such as disease or trauma,resulting in a large amount of bone loss accompanied by infection.It is the most serious complication after open fractures or orthopedic surgeries,and its pathological process includes two aspects:bone infection and bone loss.Staphylococcus aureus（S.aureus）is the main pathogen of IBD.In the process of chronic infection of bone defects,S.aureus multiplies in large numbers in the affected part of bone tissue,inhibits the activity of osteoblasts（OB）,increases the activity of osteoclast（OC）,and leads to bone reconstruction obstacles.IBD is a common orthopedic disease in canine clinical practice.The traditional treatment methods for IBD mainly use antibiotics to control infection and surgical bone grafting to promote bone recons truction.However,due to factors such as low local blood concentration of antibiotics leading to repeated infections and rejection of bone grafting materials,secondary or multiple surgeries are required,which poses great challenges to the clinical treatment of IBD.Therefore,a drug formulation that can control bone infection and promote bone reconstruction has become an urgent problem in the treatment of canine IBD.Chitosan thermosensitive hydrogel（CS/β-GP）is a drug delivery carrier,which can stably control the drug concentration at the targeted site for a long time,and has the characteristics of anti infection,promoting bone reconstruction and low side effects.It is used as the raw material of tissue engineering framework;Berberine（BBR）has antibacterial,anti-inflammatory,and bone repair promoting effects;The development of chitosan thermosensitive hydrogel drug carrier system loaded with BBR is expected to break through the difficulties in clinical treatment of canine IBD.Pharmacological effects of BBR based antibacterial,anti-inflammatory,and bone repair promotion,as well as injectable CS/β-GP thermosensitive hydrogel has good drug release and bone tissue engineering characteristics.This study focuses on"berberine chitosan thermosensit ive hydrogel can treat infectious bone defects in canine",and plans to carry out the following studies:（1）Preparation of BBR/CS/β-GP thermosensitive hydrogel,and evaluate its physical and chemical properties（p H value,surface morphology observation,rheological property,blood compatibility,biocompatibility,swelling performance,functional groups and chemical bond）,slow release effect（in vivo/in vitro slow release）and bacteriostatic effect（S.aureus strain culture,S.aureus concentration curve formulation,minimum inhibitory concentration）,to evaluate the performance of BBR/CS/β-GP thermosensitive hydrogel,and the suitable dosage of for the treatment of canine IBD was screened out.（2）Establish a canine IBD model induced by S.aureus.Divided into bone defect group,IBD group,and IBD+BBR/CS/β-GP thermosensitive hydrogel.The antibacterial,anti-inflammatory,bone repair and bone repair effects of BBR/CS/β-GP thermosensitive hydrogel on canine IBD were evaluated.Therapeutic effect of BBR/CS/β-GP thermosensitive hydrogel on S.aureus induced IBD in canines.The experimental results are as follows:（1）Preparation of the BBR/CS/β-GP thermosensitive hydrogelSuccessfully prepared BBR/CS/β-GP thermosensitive hydrogel,the gel system:CS concentration is 2%,β-GP concentration is 56%,acetic acid concentration is 0.1 mol/L,The volume ratio of CS andβ-GP is 7:3.（2）Identification of the BBR/CS/β-GP thermosensitive hydrogel（1）The gel time of CS/β-GP thermosensitive hydrogel was 4 min24 s±6 s,p H was 7.36±0.009.The gel time of BBR/CS/β-GP thermosensitive hydrogel was 4 min48 s-5 min22 s,p H was7.37-7.44.The p H value of hydrogel in each group was within the physiological p H range of canine.（2）CS/β-GP and BBR/CS/β-GP thermosensitive hydrogels all present a three-dimensional configuration,with uniform pore size and water retention effect.（3）CS/β-GP and BBR/CS/β-GP thermosensitive hydrogels have good temperature sensitivity,The phase transition temperature of CS/β-GP thermosensitive hydrogel is 37.16℃,the phase transition temperature of 250μg/m L BBR/CS/β-GP thermosensitive hydrogel is 37.56℃.With the increase of the shear rate,the shear stress of the hydrogel increases in a continuous smooth curve,indicating that the hydrogel can generate stress under external force.With the increase of the shear rate,the viscosity of the hydrogel decreases rapidly,which is shear rate dependent and conforms to the characteristics of the viscosity curve of non Newtonian liquids.（4）In the in vitro hemolysis test,the BBR/CS/β-GP thermosensitive hydrogel was mixed with canine erythrocyte suspension for 4 h without hemolysis;the hydrogel was co-cultured with canine erythrocytes,and the intact erythrocyte morphology was observed by scanning electron microscopy,indicating that the hydrogel can coexist with erythrocytes and has good hemocompatibility.（5）The in vitro compatibility test showed that 250μg/m LBBR/CS/β-GP thermosensitive hydrogel inhibited the activity of MC3T3-E1 cells in mice,BBR/CS/β-GP thermosensitive hydrogel at a concentration of 200μg/m L or less was a safe dose with good biosafety.When the canine were implanted with 200μg/m L BBR/CS/β-GP thermosensitive hydrogel,no significant edema,hemorrhage,rupture and hydrogel exposure were observed with the naked eye;histology showed normal growth of muscle tissue around the hydrogel.The animals showed no damage to the heart,liver,lung,spleen and kidney,indicating that the local administrat ion of 200μg/m L BBR/CS/β-GP thermosensitive hydrogel was safe for canine.（6）The dissolution rate of each group of hydrogels gradually decreased with the increase of test time,and the initial dissolution rate could reach about 200%,which indicated that t he hydrogels in this test had good water absorption performance.（7）BBR/CS/β-GP thermosensitive hydrogels have-OH,-COOH,-PO₄^2-and-NH₃⁺functional groups.（8）The cumulative release rate of BBR/CS/β-GP thermosensitive hydrogel can reach 70%-80%after 24 h of extended release in vitro.The peak drug concentration was reached after 7 d of in vivo extended release,and the drug remained at a certain concentration and below the safe concentration after 28 d of extended release.（9）The S.aureus concentration profile was successfully developed,and the MIC of BBR/CS/β-GP thermosensitive hydrogel for S.aureus was 150μg/m L.The above results showed that the physicochemical properties,slow release effect and bacterial inhibitory effect of BBR/CS/β-GP thermosensitive hydrogel were in accordance with the conditions of drug slow release carriers,and 200μg/m L BBR/CS/β-GP thermosensitive hydrogel was a safe dose for canine,and the concentration dose of 150μg/m L was suitable for subsequent canine IBD treatment trials.（3）Therapeutic effect of BBR/CS/β-GP thermosensitive hydrogel on IBD in canine（1）IBD modeling after 7 days of operation,the canine swelled around the wound and exuded purulent secretions,and a large number of colonies were seen in the bacterial culture of the purulent secretions.X-ray observation of bone destruction around the IBD model,patchy bone destruction areas within the cancellous bone,significant thickening of the local periosteum,and disorganization of the bone marrow mass.The morphological observation of bone tissue showed that inflammatory cell infiltration,bone loss and reticular structure destruction occurred in th e bone of the IBD model.（2）The reduced concentration of BBR remaining in the operative area of canine with IBD,the bacterial culture of purulent secretions identified as S.aureus,and the reduced number of S.aureus in the treated group compared with the IBD group indicated that the BBR/CS/β-GP thermosensitive hydrogel had an antibacterial effect on canine IBD（3）Compared with the IBD group,canine in the treatment group showed reduced leukocyte count,neutrophil percentage and fractionated neutrophil count,reduced CRP concentration,reduced IL-1βgene expression and increased IL-10 gene expression,indicating that BBR/CS/β-GP thermosensitive hydrogel has anti-inflammatory effects on canine IBD.（4）Compared with the IBD group,the treated canine showed accelerated callus growth,increased bone density.Normal bone tissue microstructure,increased collagen and muscle fibers in bone tissue,significantly increased ALP-positive cells,and significantly decreased TRACP-positive cells in treatment after 90 days of operation,indicating that BBR/CS/β-GP thermosensitive hydrogel has a bone repair effect on canine IBD.（5）Compared with the IBD group,serum Ca and P levels were restored to equilibrium,BGP levels and B-ALP activity were significantly increased,TRACP-5b and Cath-K activities were significantly decreased,and Col I,OPG,RANKL and RANK gene expressions in bone tissue were significantly increased in treatment after 90 days of operation,indicating that BBR/CS/β-GP thermosensitive hydrogel has a bone repair effect on canine IBD.In summary,this study successfully prepared BBR/CS/β-GP thermosensitive hydrogel,and the in vitro test showed that 200μg/m LBBR/CS/β-GP thermosensitive hydrogel is a safe dose for canine,and 150μg/m LBBR/CS/β-GP thermosensitive hydrogel can effectively treat S.aureus-induced IBD in canine through antibacterial,anti-inflammatory and bone repair.The results of this test can lay the theoretical foundation and test basis for the resolution of IBD.