The Effect Of Gukang On Expression Of Collagen Type Ⅰ,Ⅱ And CTGF In The Healing Process Of Osteoporositic Fracture

Objective To detect expression of collagen type â… ,â…¡ and CTGF in the healing process of osteoporositic fracture , and to explore the effect of gukang on expression of collagen type I , II and CTGF, then to explore the healing pathomechanism of osteoporotic fracture and to provide a new strategy for prevention and cure of osteoporotic fracture,by establishing animal models of osteoporotic fracture and callus culture system in vitro.Method 80 6-months-old SD male rats were selected, all made into closed fracture, and were distributed randomly into four groups,including blank group (group A), gukang-cured one (group B), testicle-ablated group(group C), and testicle-ablated with gukang-cured group (group D),each group 20 rats. After good narcosis by celliac injection with 10% hydronium about 0.33ml per 100g.cut open the rats'peritonium and vas deferens and separated dividedly testicles and epididymis of each side. Removed the testicles to simulate osteoporotic animal models, and then simulated closed fracture model using fracture-caused device. Thus came into osteoporotic closed fracture animal models. Accoding to the groups divided, rats in group B and group D were drenched with gukang liquid 10g per kg; while in group A and group C were drenched with the same volume isotonic Na chloride. All were executed at the 3,7, 14, 21 day after operation. Took out the callus in sterile circumstance, floated in the antibiotic solution (gentamicin sulfate or streptomycin) plus PBS 2 times, ablated each callus into slices about sizes of 1mm, then put them into tissue culture plate with 24 holes, each contained BGJb culture media about 1ml. And cultivated them under the condition composed of 37oC, 5% CO2 atmophere for 24 hours, then changed the culture fluid for the first time. Three days later, took out the callus and kept them frozen in LN. Tree aspects were detected: first, to observe the variance and differentiation of callus disposed with different measurements at different phases by chemistry staining, which included routine paraffin section, deparaffin and dehydration, and HE and MASSON staining. Secondly to detect expression of collagen type â…  ,â…¡I with immuno- histochemistry. Thirdly to detect respectively expression of mRNA and proteins of CTGF through RT-PCR andWstemblot.Results (l)Model: closed fracture models were made good, among them middle-fracture about 93.8%, transverse fractures 91.3%; (2)The state of callus growth at different phases and callus culture in vitro: after succeeding in setting up callus culture system, no contamination and tissue necrosis phenomena were found. In the first phase (the third day postoperation), there were hematoma and fibrous tissue connected in the fracture gap, no obviously difference found in each group. In group B hematoma were detected smaller, while callus culture in vitro grew more productively. At the second phase, we found transparent fibrocartilages connection in group A, B and D, like bamboo node, texture softly. Whilst the volume in group B was bigger than in other groups, a few pieces of violet hematoma were found in group C and D. All grew productively when cultivated in vitro, among them group B secreted more than other groups did, group A second, then came to group D and C. At the third phase, callus in group C grew as well as that in group B at the second phase; in group B and A calcification deposition were detected, which were felt hard, the callus culture growth velocity in vitro were slower than at the two former phases. At the forth phase, bony callus were detected in group B, A, D, group C changed as group A did at the third phase. With microscope we found those callus grow slower and secrete lesser. (3)HE and MASSON staining: at the third day, around the fracture place periosteom stratum germinative cells greatly proliferated, plenty of dead blood cells were arrested in the fracture gap and those fibrous reticulum; A great deal of inflammatory cells gathered, including lymphocytes, granulocytes, monocytes, macrophages and labrocytes, and a good many capillaries infiltrated into the fracture gap, about were mesenchymal cells. In group B and D appeared fibroblasts, whose cellular shape flat and slim, and the organelles underdeveloped. At the 7th day, in group B and D fibrous tissue and new capillaries proliferated obviously, outside periosteom found many osteoblasts , especially in group B and D; in group A left over a few hematoma, the fracture gap were filled with fibrous tissue, osteoblasts seldom found; there were somewhat typical hematoma in group C, which were infiltrated blood cells and inflammatory cells, and few fibrous tissue were found. Periosteom stratum germinative mesenchymal cells kept on proliferating, at the same time chondrocytes and osteoblasts differentiation took place. At the 14th day, a good deal of new bony trabecula and chondral tissue connected with each other were found at the fracture ends in group B and D, where osteoblasts and chondroblasts were active; hematoma disappeared thoroughly in group A, fibrous tissue proliferation were active, a good deal of osteoblasts came into being ,and so were some chondral tissue; in group C there was obvious fibrous tissue proliferation, and chrondrocytes and osteoblasts gathered around the fracture ends and then produced cartilage matrix and osteoid;at the 21th day, osteogenesis and chrondrogenesis strengthened, and chrondrocytes began degeneration, apotosis, while osteoclasts absorbed cartilage matrix andosteoid; in group B and D osteoblasts began to change into osteocytes and then were embedded by calcified bony matrix. Osteocytes had normal shape, in which bony couldaliculi were connected with matrix, chrondrocytes hardly found. Osteoclasts' function were active in group A and C, the reductus of cell membrane typical, many cytolysosomes found in cytoplasm, still there existed plenty of chrondrocytes of apomorphosis and necrosis embedded in caldified chrondral matrix, appearing karyopycnosis, chrondrocytes lacuna expanded, and vacuoles appeared in cytoplasm. In group B and D bony trabecula were well distributed in size and allayed in the same direction, and possessed good refraction under microscope. As to group A and C bony trabecula allayed in disorder, and refracted unevenly. (4)detection of expression of collagen type â… ,â…¡. At the 3th day, of all callus collections staining for collagen type â… ,â…¡ was negative; at the 7th day, in group A staining for collagen type â… ,â…¡ in the fracture gap was negative, while in the stratum germinativa around, staining for those secreted from mesenchymal cells transverted into chrondrocytes were weakly positive; staining in group B positive, while in group D staining for the collections were partly negative, weaker than in group A. Mostly in group C were negative, in the surrounding cartilage matrix and os cartilaginea fields staining were powerful positive, but staining for collagen type â…¡ around chrondrocytes expressed negative, singular positive and double positive; While in group B staining appeared positive or powerful positive, and in group D negative or singular positive. We found expression of collagen type â…  were weaker at the first week than at the 3th week after fracture, but expression of collagen type â…¡ were more powerful significouldtly at the 2th week than at the 3th week. Compared with group A, C and D, expression of collagen type I were more powerful in group B whether at the 2th week or at the 4 week; while expression of collagen type II weaker. (5) detection of expression of mRNA and proteins of CTGF: by employing RT-PCR technique to detect expression of CTGF gene, analysed the condition of gene expression from the view of nuclear acid and of proteins level combined with applying westernblot proteins technique to detect proteins produced from mRNA . Usually we may get more definite result of gene expression and demonstrate the condition of gene expression more detailedly and directly when analysized two layer of nuclear acid and proteins. The experiment results showed .expression of mrna and proteins appeared weak in the first phase, powerful in the 2th phase, then began to weaken gradually in group A; in group B expression appeared in advance, wheras appeared more powerful in the same phase; in group C, there were no expression in the first phase, and expression in the 2th and 3th phase were as like those in the 1th and 2th phase in group A, but weaker, the condition of expression in grouop D appeared between group A and group C.Conclusion According to the experiment results, our understanding of the healing patho-mechanism of osteoporotic fracture and review of a series of research of Chinese herbal Gukang,...