Study On Expression Of Bone-related Genes In In Vivo Tissue Engineering Bone

Because they have good osteoinductivity, HA/β-TCP ceramics with special chemical constituent and physical structure are often used as scaffold in in vivo bone tissue engineering. Studies have shown that the induced bone in HA/TCP ceramics is morphologically and histologically similar to mature bone. But until now, there is little information on gene expression of in vivo tissue engineering bone. Ideal in vivo tissue engineering bone induced by HA/TCP ceramics should have similar chronological order of gene expression to naturally healing bone. The aim of the present study is to evaluate the in vivo tissue engineering bone constructed with osteoinductive HA/TCP ceramics at both gene and protein levels by detecting type I collagen, ALP, BMP4 mRNA and ALP activity, OC protein, as compared to gene expression in naturally healing bone.The present study consisted of two parts. (1) Construction of in vivo tissue engineering bone and naturally healing bone. 36 porous HA/TCP ceramic cylinders without any addition of living cells or growth factors were implanted into the dorsal muscles of 6 dogs for 1, 2, 4, 8, 12 and 24 weeks. As control, bone defect was created at mandibular alveolus for naturally healing bone at the same time. (2) Detection of bone-related genes in in vivo tissue engineering bone at mRNA and protein level. Three cylinders of in vivo tissue engineering bone and half of naturally healing bone were used for total RNA extraction and reverse transcription, mRNA expression for type I Collagen, ALP, BMP4 and GAPDH were detected by real-time PCR method. The other specimens of two kinds of bones at week 1, 2, 4, 8 and 24 were pulverized. After quantitation of small protein, ALP activity was detected using method recommended by IFCC, while OC protein was quantified using enzyme linked immunosorption assay.The results showed that the tested genes were expressed in both in vivo tissue engineering bone and naturally healing bone from week 1 to week 24 but to a different degree. At mRNA level, the expression peak time of type I Collagen and ALP of in vivo tissue engineering bone appeared later and the two genes' maximum expression was lower. The gene expression level of BMP4 of in vivo tissue engineering bone was always significantly higher than that of naturally healing bone throughout the experimental period. At protein level, the expression peak time of ALP activity and OC protein of in vivo tissue engineering bone appeared later and their maximum expression was lower. However, the tested genes of two kinds of bones shared a similar expression tendency which was finally maintained at a relatively lower level.The results indicated that the in vivo tissue engineering bone is formed later and fewer, and its calcification is not as good as that of naturally healing bone. However, the two kinds of bones shared similar gene expression tendency, and the in vivo tissue engineering bone did neither grow uncontrollably nor disappear during the tested period.