Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1.

To treat bone defects, tissue-engineering methods combine an appropriate scaffold with cells and osteogenic signals to stimulate bone repair. Mesenchymal stem cells (MSCs) derived from adult bone marrow are an ideal source of cells for tissue engineering, in particular for applications in skeletal and hard tissue repair. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation. However, the effects of Cbfa1 on MSCs in vitro and in vivo have not been well characterized. In this study, we found that MSCs modified genetically to express Cbfa1 promoted the healing of segmental defects of the radius in rabbits. First, osteogenic differentiation of MSCs transfected with an adenovirus encoding Cbfa1 was demonstrated. Expression of mRNA from a number of osteoblastic marker genes, including osteocalcin, osteopontin, and type I collagen, was detected. In addition, alkaline phosphatase activity and increased osteocalcin content were observed. The cells expressing the Cbfa1 gene were then combined with acellular bone extracellular matrix in a flow perfusion culture system. Finally, the cell-matrix constructs were implanted into radius defects in the rabbit model. After 12 weeks, radiographic, histological, and biomechanical analyses showed that MSCs modified with the Cbfa1 gene resulted in a significantly higher amount of newly-formed bone and rebuilding of the marrow cavity than control cell-matrix constructs. This study indicates that MSCs modified with the Cbfa1 gene can act as suitable seed cells for the regeneration of bone defects.

[Triplex-forming oligonucleotide inhibits the expression of tissue factor gene in endothelial cells induced by the blood flow shear stress in rats].

AIM: To study the effect of antiparallel phosphorothioate triplex-forming oligonucleotide (apsTFO) matching with the shear stress response element (SSRE) of tissue factor (TF) gene promoter region on the expression of TF in endothelial cells (ECs) of rat common carotid artery stenosis. METHODS: The model of common carotid artery middle segment stenosis was established by silica gel pipe loop ligation in SD rats. The mRNA expression and protein synthesis of TF, early growth response-1 (Egr-1) and specificity protein 1 (Sp1) were measured by in situ hybridization (ISH) and immunohistochemistry (IHC) technique. GT21-apsTFO, GT20-apsTFO, GT20-psTFO and FITC-labeled apsTFO, matching with the SSRE of TF gene promoter region, were designed, and intravenously injected into rats at 0.5 h before operation. TFO was detected 4 h after the operation, and the mRNA expression and protein synthesis of TF, Egr-1 and Sp1 were detected 6 h after the operation. RESULTS: There were much fluorescence in vascular tissue, especially in the nuclear of ECs 4.5 h after the injection of apsTFO. The mRNA expression and protein synthesis of TF reduced by 22% - 23% with injection of GT20-apsTFO 6.5 h after stenosis (P < 0.01) and by 10% - 11% with GT21-apsTFO at the same time (P < 0.05). The inhibition by GT20-apsTFO was stronger than that of the GT21-apsTFO (P < 0.05). The expression of TF was not inhibited by the GT20-psTFO (P > 0.05). The mRNA expression and protein synthesis of Egr-1 and Sp1 did not change in the rat treated with GT20-apsTFO, GT20-psTFO and GT21-apsTFO (P > 0.05). CONCLUSION: apsTFO could mero-inhibit the expression of TF gene but could not change the expression of Egr-1 and Sp1 protein.

Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats.

Focal ischemia in the cerebral cortex results in acute and delayed cell death in the ischemic cortex and non-ischemic thalamus. We examined the hypothesis that neurons in ischemic and non-ischemic regions died from different mechanisms; specifically, we tested whether a mixed form of cell death containing both necrotic and apoptotic changes could be identified in individual cells. Focal barrel cortex ischemia in rats was induced by occlusion of small branches of the middle cerebral artery (MCA) corresponding to the barrel cortex, local blood flow was measured by quantitative autoradiography. Cell death was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 staining 1 to 10 days after the ischemia. Electron microscopy was used for ultrastructural examination. Cell death occurred in the ipsilateral cortex 24 h after ischemia, followed by selective neuronal death in the ventrobasal (VB) thalamus 3 days later. TUNEL positive neurons were found in these two regions, but with striking morphological differences, designated as type I and type II TUNEL positive cells. The type I TUNEL positive cells in the ischemic cortex underwent necrotic changes. The type II TUNEL positive cells in the thalamus and the cortex penumbra region represented a hybrid death, featured by concurrent apoptotic and necrotic alterations in individual cells, including marked caspase-3 activation, nuclear condensation/fragmentation, but with swollen cytoplasm, damaged organelles and deteriorated membranes. Cell death in the thalamus and the cortex penumbra were attenuated by delayed administration of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-FMK). Our data suggest that TUNEL staining should be evaluated with morphological changes, the hybrid death but not typical apoptosis occurs in the penumbra region and non-ischemic thalamus after cerebral ischemia.

The zinc finger protein A20 protects endothelial cells from burns serum injury.

Burn injuries as well as skin damages are often associated with immune suppression and often cause multiple organ failures. The monolayer endothelium is vulnerable to injuries from circulating factors resulting from remote wounds. Endothelial cell activation and apoptosis can alter microvascular permeability and intensify organ damage. A20, as a physiological cytoprotective gene is essential for preventing spontaneous innate immune cell-mediated inflammation and tissue destruction. It is not known whether A20 has the function to protect endothelial cells from the effect of burns serum challenge on endothelial function in vitro. This study shows that A20 can express in endothelial cells after burns serum stimulation and inhibit endothelial cell activation and apoptosis induced by burns serum. These results suggest that A20 may be beneficial in limiting the response to burn injuries.

Low shear stress regulates monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha dependent pathway.

In regions of a vessel that experience low shear stress and reversing flow patterns, early features in the pathogenesis of atherosclerosis include the accumulation of oxidized LDL (OxLDL) and adhesion of monocytes to endothelial cells (EC). Here we investigated the hypothesis that low shear stress (2 dyn/cm2) and OxLDL are synergistic for enhanced expression of vascular cell adhesion molecule (VCAM-1) and human aortic endothelial cell (HAEC)-monocyte adhesion. This study shows low shear stress can significantly reduce IkappaBalpha levels, activate NF-kappaB, increase the expression of VCAM-1 in HAEC and binding of monocytes. OxLDL itself cannot significantly increase the expression of VCAM-1 in HAEC and binding of monocytes, but through activation of NF-kappaB and degradation of IkappaBalpha induced by low shear stress it can significantly enhance VCAM-1 expression and monocyte adhesion, over that in unmodified LDL or control. These results suggest that low shear stress can regulate monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha-dependent pathway, and that low shear stress together with OxLDL may likely play an important role in atherogenesis.

[Protective effect of exogenous bcl-2 on spinal cord motoneurons following sciatic nerve axotomy in the rat].

The aim of this study was to investigate the protective effect of exogenous bcl-2 on spinal cord motoneurons following sciatic nerve axotomy. After epineurium suturing, sense bcl-2 (Ad/s-bcl-2), antisense bcl-2 (Ad/as-bcl-2), or reporter gene lacZ (Ad/lacZ) recombinant adenovirus or NS was injected into the sciatic nerve 0.5 cm distant from the sutured point respectively in different groups. The rats were transcardially perfused with 4% paraformaldehyde on postoperative 48 h, 7 d, 15 d and 30 d respectively and the spinal cords of L4 to L6 were harvested. X-gal staining, bcl-2 in situ hybridization and immunohistochemical staining, TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining and AChE (acetyl cholinesterase) histochemical staining were used. We observed that the exogenous lacZ gene was expressed in the spinal cord of Ad/lacZ group, and sense bcl-2 significantly decreased the number of apoptotic motoneurons and the decreasing degree of AChE activity of the motoneurons in the spinal cord induced by sciatic nerve axotomy and accelerate AChE activity recovery. However, antisense bcl-2 increased the number of apoptotic motoneurons and the decreasing degree of AChE activity of the motoneurons in the spinal cord induced by sciatic nerve axotomy and prolonged AChE activity recovery. These results demonstrate that exogenous bcl-2 may protect motor neurons from injury induced by peripheral nerve axotomy.

[Y-chromosome-specific microsatellite variation in Li ethnic groups of Hainan Island, China].

OBJECTIVE: The study was conducted to reveal the distribution of genetic polymorphism of four Y chromosome specific short tandem repeat (Y-specific STR) loci in Li ethnic groups in Hainan Island, China. METHODS: Four tetranucleotide STR loci were simultaneously amplified with fluorescently labeled primers, and genotypes were determined with an automated DNA sequencer. RESULTS: Among 230 unrelated males, the alleles at the four Y-specific STR loci were composed of some complex repeat structure. 4,5,4,5 alleles were observed in loci DYS3891, DYS390, DYS391, DYS393 respectively. A set of human allele ladders for the typing of the four Y-specific STRs was obtained in Li ethnic population. Gene diversity index (D) and haplotype diversity data were estimated for the four Y-STRs. CONCLUSION: The preliminary study indicates a reference population for detecting male migration events and should be useful in population genetics and forensic applications.

[Polymorphism analysis for DYS19 and DYS287 of three Li populations in Haina Island, China].

The distribution of Y-chromosome specific microsatellite DYS19, in 289 males of three ethnic populations in Hainan Island was studied with the method of PCR followed by denatured polyacrylamide gel electrophoresis and silver staining. The three ethnic populations are Bendi Li, Qi Li and Ha Li. The results showed that four alleles (190bp, 194bp, 198bp, 202bp designated as alleles B, C, D and E respectively), were observed in DYS19, the frequency ranged from 0.660-0.854, and was predominant in three Li subgroups. The pairwise comparison with Fisher's Exact test reveals that there exists significant difference (P<0.01) in DYS19 phenotype distribution between Qi Li and Ha Li populations. The polymorphism of the Y-chromosome specific Alu insert sequence DYS287 was investigated also in three Li populations. The results demonstrated that with 150 bp product in all Bendi-Li, Qi-li and Ha-li, YAP element is absent. The preliminary study provides reference populations for detecting male migration events and for reconstructing paternal history.

Multilayer scaffold of electrospun PLA-PCL-collagen nanofibers as a dural substitute.

Dural closure after the neurosurgery can prevent postoperative complications. Although many types of dural substitute have been developed, most of them lack functional and structural characteristics compared with the natural dura mater. In this study, we used electrospinning method to fabricate a multilayer scaffold to promote dural repair. The inner layer of the scaffold that faces the brain tissue is composed of poly-lactic acid (PLA) to reduce tissue adhesion. The middle layer of the scaffold is composed of poly-ɛ-caprolactone and PLA, which provides a watertight seal. The outer layer of the scaffold contains a large amount of collagen to promote cell attachment and proliferation. The results from in vitro study and an animal model have shown that this multilayer fibrous scaffold has sufficient mechanic strength and biochemical properties to enhance dural repair. Therefore, fabrication of scaffold with multiple functional and structural layers may provide a novel approach for tissue engineering.

Piezoelectric PU/PVDF electrospun scaffolds for wound healing applications.

Previous studies have shown that piezoelectric materials may be used to prepare bioactive electrically charged surfaces. In the current study, polyurethane/polyvinylidene fluoride (PU/PVDF) scaffolds were prepared by electrospinning. The mechanical property and piezoelectric property of the scaffolds were evaluated. The crystalline phase of PVDF in the scaffolds was characterised by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). In vitro cell culture was performed to investigate cytocompatibility of the scaffolds. Wound-healing assay, cell-adhesion assay, quantitative RT-PCR and Western blot analyses were performed to investigate piezoelectric effect of the scaffolds on fibroblast activities. Further, the scaffolds were subcutaneously implanted in Sprague-Dawley (SD) rats to investigate their biocompatibility and the piezoelectric effect on fibrosis in vivo. The results indicated that the electrospinning process had changed PVDF crystalline phase from the nonpiezoelectric α phase to the piezoelectric ß phase. The fibroblasts cultured on the scaffolds showed normal morphology and proliferation. The fibroblasts cultured on the piezoelectric-excited scaffolds showed enhanced migration, adhesion and secretion. The scaffolds that were subcutaneously implanted in SD rats showed higher fibrosis level due to the piezoelectrical stimulation, which was caused by random animal movements followed by mechanical deformation of the scaffolds. The scaffolds are potential candidates for wound healing applications.

Stereoscopic study on capillary density of early brain oedema in a dog postburn model.

BACKGROUND: After severe burn, the effective circulating blood volume decreases drastically due to massive body fluid loss, and blood redistribution occurs to maintain sufficient blood supply to vital organs. Blood perfusion of brain tissue changes and the permeability of the blood brain barrier increases due to ischaemia and hypoxia, which results in brain oedema. The goal of this study was to explore the changes of cerebral blood flow during the brain oedema at the early stage of severe burn. METHODS: Twenty-six dogs were randomly divided into control and 6, 12, 18, and 24 PBH groups. The manifestation of MRI and histopathology, changes of brain water content were investigated; the shapes and distribution of the cerebral capillaries were observed with the endogenous AKP histochemical staining method and image analysis technique. The volume, surface, and length fractions of cerebral capillaries were tested and analysed with a stereographic method in each group, respectively. RESULTS: The earliest changes of cerebral oedema were found at 12 PBH with MRI, which showed the brain swelling as characteristic of cerebral morphological changes. The decrease of SIR on T(1)WI was not observed until it was above 10%. Signal of T(2)WI increased for 8.29% at 24 PBH. Histological changes were observed as early as 6h after burn, accompanied by swelling of endothelial cells and peri-vascular astrocytes, vacuolation took place in neurons at 12h after burn, necrosis of different degrees in capillary endothelium, neurons, and axons. Increase in cerebral water content was noted at 6h postburn, and it was the most marked in 24 postburn group.The distributional density of capillaries became thicker at 6h and 12h postburn, the shapes were normal. The capillaries became sparser at 18h, and almost disappeared from view, only a few ends of capillaries in the shape of vine were seen at 24h postburn. The percentages of capillary volume, surface, and length fractions were increased at 6h and 12h, but decreased at 18h and reached the lowest point at 24h postburn (P<0.05). CONCLUSION: We suggest that the changes of cerebral blood flow might play an important role in the pathogenesis of brain oedema in the early stage of severe burn.

[Expression and distribution of xenoantigen alpha-Gal in intervertebral disk of Chinese banna minipig inbred line].

OBJECTIVE: To investigate the expression and distribution of xenoantigen in intervertebral disk of Chinese banna minipig inbred line, and to study the availability of xenograft transplantation of intervertebral disk. METHODS: Samples of intervertebral disk were collected from six Banna pigs of 8 to 11-month-old. The fixation, embedment and slice were performed. alpha-Gal specific binding lection (BSI-B4) were used as affinity reagents and affinity-immunohistochemistry assays (SABC methods and DAB stain) were conducted to detect the expression and distribution of xenoantigen (alpha-Gal). RESULTS: alpha-Gal was found in chondrocyte cell and chondrocyte-like cell in intervertebral disk which have the positive yellow-stained particulate aggradation. There was no stain in the matrix, elastic fiber and collagen fiber. CONCLUSIONS: The distribution of xenoantigen is locally in the tissue of intervertebral disk and its expression is weak. This suggests that the intervertebral disk of Banna pig may be alternative donor for xenotransplantation.

[Experimental study on kidney anatomic structure of banna minipig inbred-lines for xenotransplantation].

OBJECTIVE: To explore the kidney anatomic structure of banna minipig inbred-lines, and to provide data for kidney xenotransplantation. METHODS: The fresh and infused kidneys of banna minipig (including the vessel and the ureter) were checked by anatomic microscope and vernier caliper in original location and away body. The tissue structure was observed by HE stain. RESULTS: The structure of kidney of banna minipig inbred-lines (including the vessel and the ureter) are similar to that of human being. The fascia propria of kidney is divided into three layers including capsula fibrosa, capsula adipose and fascia renalis. The thickness of cortex renalis is (20.0 +/- 2.4) mm. The average diameter of renal artery is 5.1 mm and is similar to that of human being. All the kidneys of banna minipig inbred-lines have a single branch renal artery. The diameters of left and right ureters are 5.1 mm and 4.7 mm, respectively. CONCLUSION: The kidney of banna minipig inbred-lines is an ideal replacement of human kidney for xenotransplantation.

[Anatomy of heart in banna mini-pig inbred-lines].

OBJECTIVE: To observe the heart anatomic and histological structure of the Banna mini-pig inbred-lined and to provide the morphological data for heart xenotransplantation and breeding transgens pig. METHODS: Ten Banna mini-pigs (12-18 months old) were affused and fixed by common coratid artery. The heart were observed and measured by gross anatomy and histology. RESULTS: There were many similarities between the Banna pig heart and the human heart in anatomy and histology. However, the following differences were observed in the Banna pig heart: 1. Azygos vein directly drew into right atrium cordis. 2. The intercalated disk of cardiac muscle was less than that of human. 3. The Purkinje's fibre was bigger than that of human. CONCLUSION: On the morphology and histology, the structure of Banna pig heart is similar to the heart of human being. It is possible that Banna minipig heart becomes organ donors for xenotransplantation.