Properties of chitosan-collagen sponges and osteogenic differentiation of rat-bone-marrow stromal cells.

The aim of this study was to further investigate effects of a combined chitosan and collagen matrix on osteogenic differentiation of rat-bone-marrow stromal cells (BMSCs), including analysis of the physical and mechanical properties of the sponges. There were 4 study groups: collagen, chitosan, 1:1 chitosan-collagen and 1:2 chitosan-collagen sponges. Chitosan-collagen sponges were fabricated using the freeze-drying technique. BMSCs were seeded on the sponges and cultivated in mineralized culture medium for 27 days. Attachment and growth of cells on the sponges were examined under a scanning electron microscope. Alkaline phosphatase activity and levels of osteocalcin were monitored. Tests of swelling, collagenase and lysozyme enzymatic degradation, and mechanical strength were performed. The BMSCs attached successfully to the structure of the sponges, and expression of ALP and osteocalcin on collagen and chitosan-collagen composite sponges was greater than on chitosan sponges. All sponges showed a high degree of water uptake. Chitosan and chitosan-collagen sponges showed a higher resistance to enzymatic degradation than collagen sponges. A 1:1 chitosan-collagen sponge demonstrated the highest compressive strength. Combined chitosan-collagen matrixes promoted osteoblastic differentiation of BMSCs, and improved the mechanical and physical properties of the sponges.

Growth and differentiation of mouse osteoblasts on chitosan-collagen sponges.

The aim of this study was to investigate the effects of collagen on the microstructure and biocompatibility of chitosan-collagen composite sponges fabricated by a freezing and drying technique. The study was categorized into four groups: Group I: collagen; Group II: chitosan; Group III: 1:1 (by wt) chitosan-collagen and Group IV: 1:2 (by wt) chitosan-collagen sponges. A mouse osteoblast cell line, MC3T3-E1, was cultivated on the sponges in a mineralized culture medium for 21 days. Microstructure of scaffolds and growth of cells on the sponges were examined using scanning electron and confocal laser scanning electron microscopes. Pore size was analysed from scanning electron microscope images using Image-Pro Plus image analysis software. Cell viability (MTT assay), alkaline phosphatase activity and levels of osteocalcin and calcium were monitored every 3 days and on days 15 and 21, respectively. It was found that the sponges were porous with average pore sizes of 80-100 microm. A combination of chitosan and collagen matrixes created a well defined porous microstructure and biocompatible scaffolds. Chitosan-collagen composite sponges promoted growth and differentiation of osteoblasts into the mature stage. To optimize application of the composite sponges in bone regeneration, the fabrication process must be improved to increase the pore size of the scaffolds.

Skeletal stability following maxillary impaction and mandibular advancement.

The purpose of this study was to retrospectively evaluate the stability of combined Le Fort I maxillary impaction and mandibular advancement performed for the correction of skeletal Class II malocclusion. Twenty-nine patients, mean age 22.6 years, underwent bimaxillary surgery with rigid internal fixation. Standardised cephalometric analyses were performed using serial lateral cephalometric radiographs. The post-surgical follow-up was a minimum of 12 months, with a mean of 25.2 months. The maxilla was impacted by a mean of 4.3 +/- 3.3 mm, and horizontally advanced by a mean of 2.6 +/- 2.3 mm. The results demonstrated that the maxilla tended to move anteriorly and inferiorly but this was not significant in either horizontal or vertical planes (P > 0.05). The mean advancement of the mandible, at menton, was 10.7 +/- 5.6 mm, and in 14 cases (48.2%) menton was advanced greater than 10 mm. In 34.7% of the patients the mandible underwent posterior movement between 2 and 4 mm. In the vertical plane, gonion moved superiorly by a mean of 2.7 +/- 3.6 mm which was significant. Significant mandibular relapse was found to have occurred in five female patients, with high mandibular plane angles who had undergone large advancements of greater than 10 mm. In conclusion, the majority of patients undergoing bimaxillary surgery for the correction of skeletal Class II malocclusions maintained a stable result. However, a small number of patients, exhibiting similar characteristics, suffered significant skeletal relapse in the mandible secondary to condylar remodelling and/or resorption.

Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study.

Recent clinical reports suggest that the application of an autologous blood plasma enriched with thrombocytes by centrifugal concentration (platelet-rich plasma: PRP) can enhance the formation of new bone. There are very fewin vitro or in vivo studies published on the efficiency of PRP. In this project a three dimensional cell culture system was used to compare PRP and rhBMP-2 in vitro. Marrow derived bone forming cells from Spraque-Dawley (SD) rats were seeded on porous collagenous carriers (d=5mm, h=3mm) at a density of 4 x 10(4) cells/carrier and exposed to different concentrations of PRP (platelet counts from 2.5 x 10(8)-1.6 x 10(7) platelets/culture), rhBMP-2 (300 ng) or plasma poor in thrombocytes (platelet-poor plasma, PPP). Cultures without additional supplements were used as controls. During a culture period of 21 days cell proliferation, alkaline phosphatase activity (ALP) and calcium content (days 18, 21) were measured in 3 day intervals.PRP showed a dose dependent stimulation of cell proliferation, while reducing ALP activity and calcium deposition in the culture. BMP-2 led to an opposite cell response and induced the highest ALP activity and mineral deposition. These data suggest that PRP inhibited osteogenic differentiation of marrow derived pre-osteoblasts in a dose dependent manner. PRP is not a substitute for BMP-2 in osteogenic induction.

Culturing primary human gingival epithelial cells: comparison of two isolation techniques.

BACKGROUND: Cultured epithelial cells offer many potential clinical applications. There have generally been two techniques that have been used to cultivate oral keratinocytes, which include the direct explant technique and the enzymatic method. Little work has been done comparing these two techniques and their capacity to isolate and cultivate oral keratinocytes. OBJECTIVES: The objectives of this study were to (1) investigate the difference in the percentage of keratinocyte isolation between the direct explant technique and the enzymatic method of human gingival epithelial cell culture and (2) to examine the effect of age and sex of the subjects providing the tissue samples on (a) the success in cultivation and (b) the growth patterns of gingival keratinocytes. MATERIAL AND METHODS: Gingival tissue was obtained from healthy human subjects and was used for keratinocyte isolation using the direct explant technique or the enzymatic method. Epithelial cell cultures from each of the two culture techniques were selected randomly for flow cytometry analysis for cell expression of vimentin and cytokeratin. Growth rate assays were also conducted. RESULTS: The success rate for cultivation from the direct explant technique was higher (82%) than in the enzymatic method (57.9%). The success rate of both methods was not significantly associated with either age or sex of the subjects providing the tissue. From flow cytometry, the average percentage of cells that was positive to anti-pan cytokeratin was nearly the same for both methods at about 97%. It was noted that the cells from the enzymatic method gave significantly higher percentages of cells that were positive to anti-pan cytokeratin only. CONCLUSION: Both the direct explant technique and the enzymatic method can be used for isolating and culturing human oral keratinocytes. The direct explant technique appeared to be more successful in culturing human oral keratinocytes than the enzymatic method, although there were limitations found with both methods. The age and sex of the subjects providing the gingival samples did not appear to be a factor influencing the success rate in culturing the keratinocytes. However, contamination by oral microbiological flora from the gingival tissue samples remained an ever present problem. Further studies are needed in the investigation of clinical applications of these two epithelial cell isolation methods.

Anterior open-bite malocclusion: stability of maxillary repositioning using rigid internal fixation.

A retrospective cephalometric study was performed to investigate the stability of 37 non-growing anterior open-bite cases using mini-plate rigid fixation. The sample was divided into two groups: Group A: maxillary repositioning alone (17 cases) and Group B: bimaxillary surgery (20 cases). Tracings were performed pre-operatively (T1), immediately post-operatively (T2) and at a minimum of one year follow-up (T3) (12-90 months). In Group A, the maxilla was advanced (3.8 +/- 2.8 mm, p < 0.01) and superiorly repositioned at PNS (2.8 +/- 2.3 mm, p < 0.001). In Group B, the maxilla was advanced (3.5 +/- 3.0 mm, p < 0.01) and superiorly repositioned at PNS (3.7 +/- 1.8 mm, p < 0.001); and the mandible (11.7 +/- 3.8 mm, p < 0.001), with no significant change in the vertical plane (p > 0.05). Late relapse due to condylar remodelling or resorption was found as a cause of large horizontal relapse (8.0 < x < 14.0 mm) in three cases (15%), the amount being associated with the amount of operative advancement (r = 0.7, r-sq = 40%, p < 0.01). It was concluded that the correction of anterior open bite by posterior repositioning of the maxilla using rigid fixation is a stable procedure during the follow-up period, and that in bimaxillary cases, post-operative stability depends largely on the stability of the mandibular advancement, which in turn relates to the amounts of advancement, the pre-operative anterior open bite and the mandibular plane angle.

Fibrodysplasia ossificans progressiva and associated osteochondroma of the coronoid process in a child.

The article reports the occurrence of osteochondroma in a fibrodysplasia ossificans progressiva patient. A 5-year-old boy presented with limited mouth opening and firm swelling of the right zygomatic complex area. The boy had bilateral hallux valgus of the great toes and heterotopic endochondral ossification of facial and neck regions. Associated osteochondroma of the coronoid process and aggressive heterotopic ossification of masticatory and neck muscles were found in response to traumatic injuries. Natural and clinical histories of fibrodysplasia ossificans progressiva were reviewed. An early diagnosis and avoidance of factors that aggravate ossification are key factors in reducing the expected degree of physical disabilities of patients. An early recognition of congenital skeletal deformities, early detection of abnormal ossification, and awareness of the disease by the involved physicians are important factors in the early diagnosis of the disease and in reducing any unnecessary trauma. Bone scintigrams and CT scans are effective noninvasive tools for an early detection of ossification and for monitoring the progression of the disease. Further investigation of its pathogenesis at a molecular level is important to understand better the nature of the disease and to develop an effective treatment protocol.