Morphological changes in the midface of Beagle dogs treated by trans-sutural distraction / Cambios morfológicos en la cara media de perros Beagle tratados por distracción trans-sutural

Trans-sutural distraction is a biological process that induces the formation of new bone and changes the position of bone by pulling on growing suture under the action of external forces. Currently, therapy to midfacial hypoplasia treated by trans-sutural distraction has been applied. In this study, Beagle dogs were selected as experimental animals, and a traction device designed by ourselves was applied to Beagle dogs to simulate the treatment process of trans-sutural distraction in human face, so as to provide a basis for the subsequent research on the related mechanism of trans-sutural distraction. The objective is that the animal model can provide the basis for the follow-up study of transsutural distraction. 45 month beagle dogs were randomly divided into two groups 3 in experiment group and 3 in control group. Implant nails were implanted as the bone marker in the bilateral zygomatic temporal suture, zygomandibular maxillary suture and palatine transverse suture in experimental group. The traction of the maxilla was carried out by the external cranial traction frame with canine fossa as bearing point, 800g force each side, elastic traction for 15 days. The control group only implanted the implant nail as the bone marker on both sides of the bone suture. The distance between two implant nails was measured by vernier calipers and X-ray examination, compared with preoperative and postoperative changes. X-ray and cephalometric measurements were used to measure change in the cranial basal angle. HE staining was used to observe the width of the bone seams, the morphology and structure of the cells and the tissue of the new bone under the phase contrast microscope. Then descriptive statistical analysis and t-test between two independent samples are carried out for the measurement data. The experimental group had a good retention of the beagle traction frame. In the experimental group, the maxillaries of dogs were protrudent in the process of traction gradually and the occlusal relationship changed to type II malocclusion. When the traction is 15 days, the coverage distance is about 8~9 mm. Before and after the traction, the distance between landmark points indicated that the spacing between the transverse palatine suture was the largest (experimental group: 5.52±0.19 mm control group 1.31±0.06 mm P<0.05), and zygomaticotemporal suture was the second (experimental group: 3.12±0.15 mm, control group 0.73±0.04 mm, P<0.05), and zygomaticomaxillary suture was less (experimental group: 2.60±0.34 mm, control group 0.53±0.05 mm, P<0.05). The cranial basal angle was no change before and after operation (controlgroup: 32.3±1.3°, experimental group: 33.2±1.1° P>0.05. Histology showed that the collagenous fibers in the suture of the control group were denser and the osteoblasts were visible on the edge of the suture, showing osteogenic activity. The experimental group significantly widened suture (experimental group: 1209.388±42.714 µm, control group 248.276±22.864 µm, P<0.05), the number of fibroblasts increased significantly with loose collagen fiber. The direction of cell and fiber arrangement were parallel to the traction force. There were many small blood vessels and marrow cavities, and the bone trabecula around the bone suture was thin (experimental group: 23.684±3.774 mm, control group: 86.810±9.219 mm, P < 0.05), showing active osteogenic activity. The growing beagle dog can be used to establish a suture traction animal model for experimental study. In the experiment, Kirschner wire was used to penetrate the bottom plane of the piriform hole of the maxilla (about the position of the canine fossa at the back) and the traction direction was basically the same as the growth direction, and the maxilla was basically parallel and moved forward.

La distracción trans-sutural es un proceso biológico que induce la formación de hueso nuevo y cambia la posición del éste al tirar de la sutura en crecimiento bajo la acción de fuerzas externas. Actualmente, se ha aplicado la terapia para la hipoplasia de la cara media tratada por distracción trans-sutural. En este estudio, fueron seleccionados perros Beagle como animales experimentales, y un dispositivo de tracción fue instalado a los perros para simular el proceso de tratamiento de la distracción trans-sutural en el rostro humano. El objetivo fue proporcionar una base para la investigación posterior sobre mecanismos relacionados con la distracción trans-sutural. El modelo animal puede proporcionar la base para este tipo de estudio de seguimiento de la distracción trans-sutural. Perros Beagle de 45 meses de edad se dividieron aleatoriamente en dos grupos: 3 en el grupo experimental y 3 en el grupo control. Los clavos de implante se usaron como marcadores óseos en la sutura temporal cigomática bilateral, la sutura maxilar cigomandibular y en la sutura transversal palatina en el grupo experimental. La tracción del maxilar se realizó mediante el marco de tracción craneal externo con fosa canina como punto de apoyo, 800 g de fuerza a cada lado, tracción elástica durante 15 días. En el grupo control solo se implantó el clavo del implante como marcador óseo en ambos lados de la sutura. La distancia entre dos clavos de implante se midió mediante calibradores de vernier y examen de rayos X, en comparación con los cambios preoperatorios y postoperatorios. Se utilizaron mediciones cefalométricas y de rayos X para medir el cambio en el ángulo basal craneal. La tinción con HE se usó para observar el ancho de las suturas óseas, la morfología y la estructura de las células y el tejido del hueso nuevo bajo el microscopio de contraste de fase. Luego se realizó un análisis estadístico descriptivo y una prueba t entre dos muestras independientes para los datos de medición. El grupo experimental tuvo una buena retención del cuadro de tracción del Beagle. En el grupo experimental, los maxilares de los perros sobresalieron gradualmente en el proceso de tracción y la relación oclusal cambió a maloclusión tipo II. Cuando la tracción era de 15 días, la distancia de cobertura fue de aproximadamente 8 ~ 9 mm. Antes y después de la tracción, la distancia entre los puntos de referencia indicaba que el espacio entre la sutura palatina transversal era más grande (grupo experimental: 5,52 ± 0,19 mm, grupo de control 1,31 ± 0,06 mm, P <0,05), y la sutura cigomáticotemporal fue la segunda. (Grupo experimental: 3,12 ± 0,15 mm, grupo control 0,73 ± 0,04 mm, P <0,05), y la sutura cigomaticomaxilar fue menor (grupo experimental, 2,60 ± 0,34 mm, grupo control 0,53 ± 0,05 mm, P <0,05). El ángulo basal craneal no cambió antes ni después de la operación (grupo control 32,3 ± 1,3, grupo experimental, 33,2 ± 1,1 ° , P> 0,05). La histología mostró que las fibras colágenas en la sutura del grupo control eran más densas y los osteoblastos se observaron en el margen de la sutura, mostrando actividad osteogénica. En el grupo experimental se amplió significativamente la sutura (1209,388 ± 42,714 µm, grupo control 248,276 ± 22,864 µm, P <0,05), el número de fibroblastos aumentó significativamente con fibras colágenas dispersas. La dirección de la disposición de la celda y las fibras era paralela a la fuerza de tracción. Se observó gran cantidad de vasos sanguíneos pequeños, cavidades medulares, y trabéculas óseas alrededor de la sutura ósea (grupo experimental: 23,684 ± 3,774 mm, grupo control: 86,810 ± 9,219 mm, P <0,05), que mostró actividad osteogénica activa. El perro Beagle en crecimiento se puede utilizar para estudios experimentales y así establecer un modelo animal de tracción de sutura. En el proceso, se usó alambre de Kirschner para penetrar en el plano inferior del foramen piriforme del maxilar (aproximadamente en la posición de la fosa canina en la parte posterior) y la dirección de tracción fue básicamente la misma que en el crecimiento.

Morphological observation of bone marrow mesenchymal stem cells under matrigel three dimensional culture conditions / Observación morfológica de células madre mesenquimales de médula ósea en condiciones de cultivo tridimensional con matrigel

SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.

RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.

[Mandibular-driven simultaneous maxillo-mandibular distraction for hemifacial microsomia with rapid prototyping technology].

OBJECTIVE: To explore the clinical application of mandibular-driven simultaneous maxillo-mandihular distraction to correct hemifacial microsomia with rapid prototyping technology. METHODS: The patient' s skull resin model was manufactured with rapid prototyping technology. The osteotomy was designed on skull resin model. According to the preoperative design, the patients underwent Le Fort I osteotomy and mandibular ramus osteotomy. The internal mandible distractor was embedded onto the osteotomy position. The occlusal titanium pin was implanted. Distraction were carried out by mandibular-driven simultaneous maxillo-mandihular distraction 5 days after operation. RESULTS: The distraction in five patients was complete as designed. No infection and dysosteogenesis happened. The longest distance of distraction was 28 mm, and the shortest distance was 16 mm. The facial asymmetry deformity was significantly improved at the end of distraction. The ocelusal plane of patients obviously improved. CONCLUSIONS: Rapid prototyping technology is helpful to design precisely osteotomy before operation. Mandibular-driven simultaneous maxillo-mandibular distraction can correct hemifacial microsomia. It is worth to clinical application.

[Quantitative analysis of craniofacial skeleton asymmetry by three-dimensional computed tomography].

OBJECTIVE: To present a method of quantitative diagnosis of craniofacial skeleton deformities based on three-dimensional computed tomography (3D CT). METHODS: 20 cases with facial asymmetric deformities underwent 3D CT and the 3D images were reconstructed by Mimics 10.0 (Belgium). Anatomical landmarks were located and the coordinate of the landmarks obtained. Axial images of 1 patient with Romberg disease was used as representative case. The differences in the distance between the right landmarks and the left were calculated and analyzed. RESULTS: The measurement results were not significantly different between two stages with an interval of 4 weeks ( P > 0.05), showing a reproducible resutls. The deviation of landmarks at facial midline increased gradually from upward to downward, reaching (2.63 +/- 0.54) mm at menton point. Paired landmarks showed asymmetry in three dimensions, especially gonion point on the left side, which was deviated 10.21 mm inward, 9.26 mm forward, 6.30 mm upward, compared to the opposite side. CONCLUSIONS: The method of 3D CT quantitative analysis can provide precise information in the diagnosis and treatment planning of facial asymmetry deformity.

[Effects on collagen I and III after transplantation of porcine acellular dermal matrix with hyaluronic acid].

OBJECTIVE: To investigate the changes of collagens I and III after the addition of hyaluronic acid in the transplantation of porcine acellular dermal matrix. METHODS: Full-thickness skin defects were created on the dorsa of Japanese white rabbits. And the rabbits were divided randomly into 3 groups: Group A (hyaluronic acid, porcine acellular dermal matrix plus thin skin autografts), Group B (porcine acellular dermal matrix plus thin skin autografts) and Group C (skin autografts). Skin biopsies were performed at Day 50 post-grafting to detect the contents of collagens I and III by histological examinations, immunohistochemistry method and Western blot. RESULTS: The areas of skin graft were (13.3 ± 1.2), (9.5 ± 0.9) and (10.0 ± 1.4) cm(2) in Groups A, B and C respectively. Group A was larger than Groups B and C(all P < 0. 01). There was no statistical difference between Groups B and C (P > 0.05). The expressions of collagen I were 1894 ± 164, 515 ± 38 and 395 ± 43 in Groups A, B and C respectively. Group A was higher than Groups B and C (P < 0.01). And the expressions of collagen III were 5411 ± 435, 874 ± 70 and 2078 ± 175 in Groups A, B and C respectively. Group C was higher than Group B and yet lower than Group A (all P < 0.01). The ratios of collagen I and collagen III in Group A (0.39) and Group B (0.59) were higher than that of Group C (0.19) (all P < 0.01). CONCLUSION: The addition of hyaluronic acid may boost the expression of collagens I and III and decrease the ratio of collagen I/collagen III. Thus it facilitates wound healing and basilar membrane remodeling and alleviates the contraction of skin transplant.

[An ultrastructure study on the palatomaxillary suture of dog expanded by NiTi-SMA].

OBJECTIVE: To explore the ultrastructure of the palate-maxillary sutures under tensile forces by transmission electron microscope (TEM). METHODS: The Suture expanders were made in NiTi-Shape memory alloy (NiTi-SMA). The maximum force was 3.5 N. Fourteen 8-month old mongrel dogs were used in the study. They were divided into three groups, (1) experimental group, (2) control group, (3) sham group. In the experiment and control groups, an 8 mm wide cleft was made by surgery. The suture expanders were fixed onto the palatine bones of the experimental group. The dogs of the experimental group were executed after 3, 7, 14, 28, 56 days of suture expansion. The change of suture tissue was examined by TEM. RESULTS: The cleft of the experiment group were closed at the ninth day of expansion. At the beginning, tissue rupture, exudation, death of fibroblasts, disruption of collagen and tear vessels were seen at the early stage of suture expansion. Then highly active functional manifestations were seen in both osteocytic and fibrocytic populations. At last, normal structure restored. CONCLUSIONS: Cell types and functional condition could be distinguished clearly by TEM. It suggests that the suture expansion should be the process of tissue repair and regeneration. The suture cells response, especially, the osteogenic response were the major factor of increasing suture width.

[Application of thoracic skin flap with multiple blood supply in repair of tissue defects and deformities in jaw and neck].

OBJECTIVE: To explore an appropriate measure to repair tissue defects and deformities in mandibulo-cervical region. METHODS: Eighteen cases with severe tissue defects and deformity in jaw and neck were repaired with thoracic skin flap with multiple blood supply system in our unit from Jan. 2006 to Nov. 2008. Anterior cutaneous branch of transverse cervical artery, intercostal branch of internal thoracic artery and lateral thoracic artery were included in the pedicles. RESULTS: All skin flaps survived, except in one patient in whom a small belb appeared at the distal end of the island flap with anterior cutaneous branch of transverse cervical artery, and it was healed after a few dressing changes. The functions and appearances were satisfactory after 6-month to 2-year follow-up, without showing secondary deformity. CONCLUSIONS: The blood supply of thoracic skin flap is abundant and constant, which is an ideal method for repair of tissue defects and deformities in jaw and neck after taking into account some factors, such as the demand of the patient, general physical condition, and the size of the defect.

[Three-dimensional finite element analysis for different directions distraction at midface].

OBJECTIVE: To investigate the biomechanical changes with different directions distraction at midface. METHODS: An anteriorly directed 500 g force was applied to the floor of apertura piriforms in different directions to the occlusal plane. Three-dimensional finite element analysis was used to evaluate the biomechanical change of craniofacial complex. RESULTS: As the force direction was moved downward, the sagittal distraction length of the craniofacial complex decreased and vertical movement changed from upward to downward. The craniofacial complex was moved anteriorly when the downward force was applied about 20-30 degrees to the occlusal plane. The forces could generate the uniform stress distribution in the craniofacial sutures and avoid counterclockwise rotation of the maxilla. CONCLUSIONS: The craniofacial complex can be effectively distracted anteriorly when the downward force is applied to the floor of aperture piriforms in direction of 20-30 degrees to the occlusal plane.

[Three-dimensional finite element analysis of craniofacial skeleton protraction at medium position].

OBJECTIVE: The purpose of this study was to investigate the biomechanical changes of midface skeleton protraction at its medium position in the craniofacial complex, using the three-dimensional finite element method (FEM). METHODS: A three-dimensional FEM model was developed from the CT scan images by the technologies of three-dimensional reconstruction, image processing and meshing. The protraction forces were applied to the following locations: the first molar, full maxillary arch, and the floor of aperture piriforms. Biomechanical changes from different position protraction were investigated by means of finite element analyses. RESULTS: Protraction forces at the level of the floor of aperture piriforms produced a more forward movement of the upper maxilla in sagittal direction. Vertical and lateral displacements were less than those in loading with teeth or denture. Compressive stress on the radix nasi decreased obviously in midface skeleton protraction at its medium position. CONCLUSIONS: Compared with traditional orthopedic protraction, midface skeleton protraction at its medium position could advance maxilla en bloc, decrease the counterclockwise rotation of the maxilla, and reduce the constriction of the anterior part of the palate.

[Suppositional sutural construction and application in the three-dimensional finite element model of craniofacial complex].

OBJECTIVE: The purpose of this study was to explore suppositional sutural construction and protraction in the three-dimensional finite element model of craniofacial complex. METHODS: Combining spiral CT scanning technology with the three-dimensional finite element method, three-dimensional FEM model of craniofacial complex was developed for analysis. In the model, craniofacial sutures were constructed through the MSC. Patran program and different mechanical properties of sutures were applied. Under the same loading condition, bone displacements of the maxilla were calculated. RESULTS: A three-dimensional FE model of craniofacial complex, comprising teeth and craniofacial sutures, was developed. The displacement of the skeletal structures with sutures was significantly larger than that without sutures. CONCLUSIONS: The construction of craniofacial sutures in the three-dimensional finite element model of craniofacial complex could improve the accuracy of finite element model, which set up good foundation for biomechanical studies of craniofacial complex.

[Construction and expression of eukaryotic expression vector containing hVEGF165 gene in rat bone marrow stroma cell].

AIM: To construct the eukaryotic expression vector containing human vascular endothelial growth factor 165(VEGF165) gene and express it in rat bone marrow stroma cells(rMSCs). METHODS: The recombinant plasmid pSP73-VEGF165 was digested with BamH I and Xho I. Then the hVEGF165 gene segment obtained was again cloned into pcDNA3.1 to construct recombinant eukaryotic expression vector pcDNA3.1-VEGF165. Then the recombinant vector was identified by enzyme digestion analysis and sequencing. The rMSCs were transformed by recombinant vector and positive clones were screened with G418. The expression of hVEGF165 gene in the transformed cells was detected by immunocytochemical staining. RESULTS: Enzyme digestion analysis and sequencing showed that target gene had been cloned into recombinant vector. The expression of hVEGF165 gene in the transformed cells had been demonstrated by immunocytochemical staining. CONCLUSION: The recombinant eukaryotic expression vector has been constructed and expressed successfully in the transformed cells. Therefore, it is possible to use the rMSCs expressing hVEGF165 gene as seed cells in the bone tissue-engineering.