Advances in biomimetic modification of materials for oromaxillofacial bone regeneration and dental implant / 华西口腔医学杂志

Oromaxillofacial hard tissue defects is still a difficult problem in clinical treatment. Regeneration of oromaxillofacial hard tissue based on tissue engineering technology has a good clinical application prospect. The functional modification of scaffolds is one of key factors that influence the outcome of tissue regeneration. The biomimetic design of biomaterials through simulating the natural structure and composition of oromaxillofacial hard tissue has gradually become a research hotspot due to its advantages of simplicity and efficiency. In this article, the biomimetic modification of biomaterials for oromaxillofacial hard tissue regeneration is reviewed, expecting to provide a new idea for the treatment of oromaxillofacial hard tissue defect.

The experimental study on porous calcium phosphate cement with bone marrow stromal cells for bone tissue engineering / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To observe the biocompatibility of new biomaterials porous calcium phosphate (CPC) and ectopic bone formation of CPC with bone marrow stromal cells (BMSCs).</p><p><b>METHODS</b>The BMSCs were cultured from Beagle dog and combined with the porous CPC with the best concentration after transfect green fluorescent protein (GFP). The adhesion and growth of BMSCs on CPC were observed under inversion, fluorescence and scanning electron microscopy. The ectopic bone formation were observed at the 8th week after CPC and BMSCs were implanted subcutaneously into nude mice.</p><p><b>RESULTS</b>When BMSCs with CPC were cultured at the 1st day, cells were climbing out from CPC with normal morphology. At the 7th day cells can be seen protruding pseudopods, secretion of matrix. Bone formation could be seen histomorphologically at the 8th week.</p><p><b>CONCLUSION</b>Porous CPC has good biocompatibility and is an ideal scaffold material for bone tissue engineering.</p>

Sequential fluorescent labeling observation of maxillary sinus augmentation by a tissue-engineered bone complex in canine model / 国际口腔科学杂志·英文版

<p><b>AIM</b>To evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex of beta-tricalcium phosphate (beta-TCP) and autologous osteoblasts in dogs.</p><p><b>METHODOLOGY</b>Autologous osteoblasts from adult Beagle dogs were cultured in vitro. They were further combined with beta-TCP to construct the tissue-engineered bone complex. 12 cases of maxillary sinus floor elevation surgery were made bilaterally in 6 animals and randomly repaired with the following 3 groups of materials: Group A (osteoblasts/beta-TCP); Group B (beta-TCP); Group C (autogenous bone) (n=4 per group). A polychrome sequential fluorescent labeling was performed post-operatively and the animals were sacrificed 24 weeks after operation for histological observation.</p><p><b>RESULTS</b>Our results showed that autologous osteoblasts were successfully expanded and the osteoblastic phenol-types were confirmed by ALP and Alizarin red staining. The cells could attach and proliferate well on the surface of the beta-TCP scaffold. The fluorescent and histological observation showed that the tissue-engineered bone complex had an earlier mineralization and more bone formation inside the scaffold than beta-TCP along or even autologous bone. It had also maximally maintained the elevated sinus height than both control groups.</p><p><b>CONCLUSION</b>Porous beta-TCP has served as a good scaffold for autologous osteoblasts seeding. The tissue-engineered bone complex with beta-TCP and autologous osteoblasts might be a better alternative to autologous bone for the clinical edentulous maxillary sinus augmentation.</p>

Fabrication of titanium dioxide nanotube array and effects of its osteoblast proliferation and alkaline phosphatase activity / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate preparation parameters of TiO2 nanotube layer with anodization, and to evaluate the osteoblast activity on TiO2 nanotube layer in vitro.</p><p><b>METHODS</b>Titanium dioxide nanotube layer was grown using anodization method. TiO2 nanotube layers with different structure were obtained by controlling the voltage and time of anodization and rinsing process after anodization. Pure titanium without anodization was used as control. Osteoblasts were cultivated on the anodizated TiO2 nanotube, and the growth of osteoblasts was then evaluated and analyzed.</p><p><b>RESULTS</b>The voltage, duration of anodization and rinsing process following anodization were key factors to affect morphology of TiO2 nanotube layer. The area of osteoblast cultured on TiO2 nanotube layer was larger than that on pure titanium. Results from methylthiazol tetrazolium (MTT) test showed that the proliferation of osteoblasts after 96 h cultivation on TiO2 nanotube layer (0.62 +/- 0.02) was significantly higher than that on unanodizated titanium (0.55 +/- 0.03, P < 0.05). Three weeks later, the alkaline phosphatase (ALP) activity on TiO2 nanotube layer [(130.8 +/- 5.1) A(405)/mg] was significantly higher than that on unanodizated titanium [(109.6 +/- 4.5) A(405)/mg, P < 0.05].</p><p><b>CONCLUSIONS</b>The structure of TiO2 nanotube layer was greatly associated with anodization voltage. The TiO2 nanotube layer had a positive effect on osteoblast behavior.</p>

An ectopic study of tissue-engineered bone with Nell-1 gene modified rat bone marrow stromal cells in nude mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.</p><p><b>METHODS</b>bMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.</p><p><b>RESULTS</b>Under current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05).</p><p><b>CONCLUSIONS</b>This study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.</p>

The establishment of conditional MT transgenic mice model / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo.</p><p><b>METHODS</b>Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two elements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene cassette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts (target) in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR.</p><p><b>RESULTS</b>The conditional transgenic vector was constructed successfully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjection. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding.</p><p><b>CONCLUSION</b>Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.</p>

The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tissue-engineering techniques combined with gene therapy have been recently reported to improve osteogenesis. In this study, tissue-engineered bone constructed by human Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) was explored in an ectopic bone formation model in rabbits.</p><p><b>METHODS</b>A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected in vitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the target gene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted to explore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined with Natural Non-organic Bone (NNB), a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x 3 mm, at a concentration of 5 x 10(7) cells/ml. They were subsequently implanted into 6 rabbits subcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.</p><p><b>RESULTS</b>pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, gene transfer efficiency reached more than 30%. Target gene transfer could strengthen the transcription of BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In the ectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in the bMSCs group was (17.2 +/- 7.1)%, and pEGFP group was (14.7 +/- 6.1)%, while pEGFP-hBMP-4 group was (29.5 +/- 8.2)%, which was the highest among the groups (F = 7.295, P < 0.01).</p><p><b>CONCLUSIONS</b>The mammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transfer efficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoted differentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection of bMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bone tissue engineering applications.</p>

The study of hBMP-4 gene modified tissue-engineered bone / 中华口腔医学杂志

<p><b>OBJECTIVE</b>Bone marrow stromal cells (bMSCs) of rabbits transferred with mammalian hBMP-4 expression plasmid were used to construct tissue-engineered bone. Gene therapy combined with tissue-engineering technique was explored to further improve osteogenesis.</p><p><b>METHODS</b>pEGFP-hBMP-4 plasmid was constructed by subcloning technique. bMSCs were then transferred with either pEGFP-hBMP-4, pEGFP plasmid by lipofectamine or left uninfected in vitro. The cells from the 3 groups were combined with natural non-organic bone (NNB) to construct tissue-engineered bones, which were subcutaneously implanted into nude mice (6 implants per group) for 4 weeks. Specimens were evaluated through histological and computerized new bone formation analysis.</p><p><b>RESULTS</b>pEGFP-hBMP-4 plasmid was successfully constructed. bMSCs could attach and proliferate on the surface on NNB. In vivo experiment showed that new bone formation in pEGFP-hBMP-4 group was higher than those of the control groups.</p><p><b>CONCLUSIONS</b>Tissue-engineered bone using hBMP-4 gene modified bMSCs might be an ideal alternative for the repair of bone.</p>

Effects of transient transfection of human BMP-4 gene on rabbit bone marrow stromal cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>Bone marrow stromal cells (MSCs) were transfected with human bone morphogenetic protein-4 (hBMP-4) gene in vitro to provide BMP gene modified cells for tissue-engineered bone.</p><p><b>METHODS</b>MSCs were cultured and transfected with pEGFP-hBMP4, pEGFP plasmids respectively or left uninfected as control. Transcription of BMP-4 gene as well as gene transfection efficiency was tested. Morphological and growth feature of the transfected cells were valued. Alkaline phosphatase (ALP), von Kossa, and Osteocalcin (OC) were tested to determine the phenotypes of osteoblast.</p><p><b>RESULTS</b>The gene transfection efficiency was 20%-30%, based on GFP expression. RT-PCR showed that MSCs had low transcription of BMP-4 that was enhanced by the gene transfer. Morphological feature of MSCs transfected with pEGFP-hBMP-4 changed but growth curves did not show much difference among the groups. In pEGFP-hBMP-4 group, ALP positive stain area and the number of calcium nodules were increased, as well as the expression of OC.</p><p><b>CONCLUSIONS</b>A high transfer efficiency of MSCs was achieved under optimized conditions. The gene transfer technique strengthened the transcription of BMP-4 and promoted differentiation from MSCs to osteoblasts. hBMP-4 transferred MSCs may serve as an ideal cell source for tissue-engineered bone.</p>

Preventive effects of cerebro cellular growth peptide on gentamycin-induced inner ear damage in guinea pigs / 中国应用生理学杂志

<p><b>AIM</b>To investigate the preventive effects of the cerebro cellular growth peptide (CCGP) on gentamycin-induced inner ear damage in guinea pigs, and to clarify its mechanism.</p><p><b>METHODS</b>The hypoacusis severity and enzymatic activity in the cochlear hair cells were examined by brainstem auditory evoked potential (BAEP) and histochemistry, respectively. The damaged hair cells was counted in three groups.</p><p><b>RESULTS</b>CCGP reduced the elevated BAEP reaction thresholds. It protected activities of mitochondrial succinate dehydrogenase and lysosome acid phosphatase in the cochlear hair cells. The number of damaged hair cells in the CCGP group was less than that in the gentamycin (GM) group.</p><p><b>CONCLUSION</b>CCGP can reduce GM ototoxicity. The mechanism may be associated with the protective activity of mitochondrial enzyme, the maintenance of lysosome intactness, energy metabolism of the cochlear hair cells, and reduction of autolysis of hair cells induced by hydrolase over flowing from lysosome.</p>