Experimental study on the bone microstructure and bone morphogenetic protein-4 expression during fracture healing in a rat osteoporotic model prepared by ovariectomy

Wei SUN; Zirong LI; Zhenming HU

ABSTRACT

BACKGROUND:

As the result of estrogen shortage due to ovariectomy,osteoporosis occursin the general and local bones, displaying bone loss and changes in bone microstructure and growth factor mRNA expression,which definitely has an important effect on fracture healing.

OBJECTIVE:

To probe into the effect of estrogen on bone microstructure and bone morphogenetic protein-4 mRNA expression and location during fracture healing.

DESIGN:

Randomized controlled study.

SETTING:

Laboratory of the Department of Molecular Biology of Yunnan University. MATERIALS This study was conducted at the laboratory of Molecular Biology Department of Yunnan University between July 1999 and July 2002. We recruited 96 healthy female SD rats of 2 months old and with the mean body mass of 160 to 200 g.

METHODS:

①Of the 96 rats that received intraperitoneal anesthesia, 48rats were subjected to bilateral ovariectomy(osteoporotic group), and the other 48 rats received sham operation (control group). One month later, bilateral tibia fracture at the middle segment was artificially made on all rats under anesthesia, and no treatment was given so as to prepare fracture healing model. Then rats of both groups were put to death for collecting callus and the surrounding parenchyma at postoperative 1, 3, 5, 7, 14, 28,56 and 112 days, with 6 rats in each time point. ② The tibia bone microstructure was observed under electron microscope. ③ The mRNA expression of bone morphogenetic protein-4 at callus and the surrounding paranchyma was detected by RT-PCR method; no probe in hybrid fluid was used as negative controls. Six in situ hybridization slices with positive expression were selected from both groups at postoperative 1 and 3 days time points, and 3 visual fields were randomly selected from each slice for observing the positive granules under 25 times field lens. MAIN OUTCOME

MEASURES:

①The tibia bone microstructure;② bone morphogenetic protein-4 mRNA expression at callus and the surrounding parenchyma.

RESULTS:

All the 96 rats entered the final results analysis. ① Observation of the tibia bone microstructure at 28 days after tibia fracture, osseous callus and ostein fibers were found arranged densely in control group. Osteocytes, small with fewer cytoplasts, were observed in osseous lacuna, but osteoclasts were found surrounding small-sized bone trabecula. In osteoporotic group, fibrous callus and collagenous fibers in bone matrix were arranged loosely, lots of big osteoblasts could be observed with osteocytes easily seen. ② Bone morphogenetic protein-4 mRNA expression at callus and the surrounding parenchyma it was less expressed in control group than in osteoporotic group at postoperative 1 to 3 days (23.714 3±5.056 8,21.714 3±5.023 8 vs 51.285 7±8.138 7,49.571 4±9.071 1, P ＜ 0.01) and the expression was mainly observed in parenchyma surrounding the fracture where callus was formed.

CONCLUSION:

Bone morphogenetic protein-4 mRNA expression is increased in osteoporotic group and is mainly observed in parenchyma surrounding the fracture, displaying a manner of regional expression. However,the formation and quality of callus matrix during fracture are obviously poorer than in control group.