脱乙酰壳多糖支架在牙周组织工程中的应用

脱乙酰壳多糖生物材料尽管具有良好的生物相容性,但其作为牙周组织工程支架材料时也有自身的局限和不足。本文就牙周组织工程、脱乙酰壳多糖、脱乙酰壳多糖牙周组织工程支架等研究进展作一综述。     

Alkaline phosphatase activity in human periodontal ligament: age effect and relation to cementum growth rate

Recently, a relationship was demonstrated between the thickness of the cementum layer in rat molars and the activity of alkaline phosphatase (ALP) in the adjoining periodontal ligament (PDL). It was the aim of the present study to investigate whether such a relationship also exists in the periodontium of man. Healthy deciduous and permanent teeth free from periodontitis were obtained from 74 patients, varying in age from 3 to 78 yr, and their PDL dissected from the middle one-third of the roots. ALP activity was measured in PDL extracts and expressed per hydroxyproline content. It was shown that ALP activity was relatively high in children. After puberty its concentration decreased to level off at about half the concentration found in the younger age groups. The activity of the enzyme in the PDL correlated positively with the yearly cementum thickness increment as calculated from data published previously.     

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目的观察苯妥英钠（PHT）对内毒素作用下人牙周膜干细胞（h PDLSCs）在牙骨质片上附着的影响。方法本研究于2012年6月至2013年2月在滨州医学院中心实验室和西安交通大学医学院中心实验室完成。利用扫描电镜和透射电镜观察h PDLSCs分别在20μg/m L PHT＋100 ng/m L脂多糖（LPS）与100 ng/m L LPS作用下在牙骨质片上的附着情况以及细胞超微结构的变化。结果与细胞培养液中加入100 ng/m L LPS比较,培养液中加入20μg/m L PHT＋100 ng/m L LPS培养72 h后h PDLSCs胞浆内细胞器更丰富,粗面内质网扩张,高尔基复合体发达,有大量的线粒体和核糖体;培养第7 d和第21 d,细胞生长更密集,伪足较多且粗大,与牙骨质片的附着更紧密,细胞周围钙盐沉积以及胶原蛋白合成更加活跃。结论 PHT可促进h PDLSCs在牙骨片上的附着和生长。     

Location of putative stem cells in human periodontal ligament

BACKGROUND AND OBJECTIVE: The origin of cells in the mature periodontium, and the location of their progenitors, are still unknown. It is also unknown whether inflammation influences the number and distribution of these cells within the periodontium. Molecules such as STRO-1, CD146 and CD44 have been identified on a variety of mesenchymal stem cells. The aim of this study was to identify and localize putative stem cells in diseased and healthy human periodontal ligament using cell-surface markers for mesenchymal stem cells. MATERIAL AND METHODS: Healthy and periodontitis-affected teeth were collected, fixed in 10% neutral-buffered formalin, decalcified and embedded in paraffin in preparation for immunohistochemistry. Antibodies against STRO-1, CD146 and CD44 were used to identify putative stem cells in the periodontal ligament. RESULTS: Putative stem cells were identified in both healthy and diseased periodontal ligament. They were mainly located in the paravascular region and small clusters of cells were also found in the extravascular region. Wider distributions of these cells were detected in sections of diseased ligament. CONCLUSION: Within the periodontal ligament of both healthy and diseased teeth, cells have been identified consistent with their identification as putative stem cells. The presence of an inflammatory reaction associated with periodontitis may enhance the number of these cells.     

Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs

Abstract The effect of a collagen gel matrix as a submembranous space-maintaining material was evaluated in guided tissue regeneration procedures. In 4 dogs, contralateral surgical circular fenestration defects, 5 mm in diameter, were produced at the midbuccal aspect of the alveolar bone in 8 maxillary canines. Removal of bone, PDL and cementum was complete. Experimental sites were filled with collagen gel and covered with collagen membranes; control sites were covered with collagen membranes and the underlying space was spontaneously filled with blood. Mucogingival flaps were repositioned. Histological and histomorphometric observations, 6 weeks post-surgery, indicated that defects covered by collagen membranes presented the most impressive regeneration with almost complete coverage of the denuded root by new cementum (98.4%) and new bone (63.2%). In the experimental defects. 83.5% coverage of new cementum with only 21.9% new bone regeneration was observed. These results suggest that collagen gel. interfered with healing by PDL and bone-derived cells in the submembranous space.     

牙周组织工程研究新进展

牙周炎是累及4种牙周支持组织的炎症性、破坏性疾病,现在包括GTR手术等传统的牙周治疗手段通常不能实现已破坏的牙周组织完全再生。近年来,大量的促进牙周再生方面的研究集中在牙周组织工程方面。本文就牙周组织工程的研究现状做一综述。     

Regenerative potential and healing dynamics of the periodontium: a critical‐size supra‐alveolar periodontal defect study

Objectives: The nature and characteristics of the newly formed periodontium obtained following regenerative procedures remain a matter of controversy. The objective of this study was to evaluate the regenerative potential of the periodontal attachment and healing dynamics as observed from the spatial distribution of newly formed cementum, periodontal ligament (PDL) and alveolar bone following optimal circumstances for wound healing/regeneration in a discriminating animal model. Material and Methods: Critical‐size, 6‐mm, supra‐alveolar, periodontal defects were surgically created in six young adult Beagle dogs. Space‐providing ePTFE devices with 300‐μm laser‐drilled pores were implanted to support wound stability and space provision in one jaw quadrant/animal. Treatments were alternated between left and right jaw quadrants in subsequent animals. The gingival flaps were advanced to submerge the defect sites for primary intention healing. Histometric analysis followed an 8‐week healing interval. Results: Healing was uneventful in all animals. The histometric analysis showed that cementum regeneration (2.99 ± 0.22 mm) was significantly greater than PDL (2.54 ± 0.18 mm, p=0.03) and bone regeneration (2.46 ± 0.26 mm, p=0.03). The wound area showed significant positive non‐linear effect on cementum (log β=1.25, p<0.001), PDL (log β=1.24, p<0.001) and new bone formation (log β=1.36, p<0.001). A high degree of concordance and significant linear relationship was observed between cementum, PDL and bone regeneration indicating that their formation virtually occurred in parallel. Conclusions: Cementum, PDL and alveolar bone virtually regenerate in parallel under optimal circumstances for periodontal wound healing/regeneration. Moreover, space provision positively influences the extent of periodontal regeneration.     

牙周膜干细胞的研究进展

牙周组织工程是牙周病治疗研究的热点,牙周膜干细胞是其关键种子细胞之一。本文就牙周组织工程的相关研究和牙周膜干细胞的来源、分离与培养、细胞表型、生物学特性、功能影响因素和分子调控进行综述,并对其面临的挑战和前景作一讨论。     

Periodontal repair in dogs: effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of alveolar bone and periodontal attachment

OBJECTIVES: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to stimulate alveolar bone and cementum formation in periodontal defects but not a functionally oriented periodontal ligament (PDL). Subcutaneous and intramuscular implants of BMP-12 have been shown to induce tendon formation and ligament-like tissue. The objective of this study was to evaluate rhBMP-12 for periodontal regeneration, in particular PDL formation. METHODS: Six young adult Hound Labrador mongrel dogs were used. Routine supraalveolar periodontal defects were created around the mandibular premolar teeth. Three animals received rhBMP-12(0.04 mg/ml) in an absorbable collagen sponge (ACS) carrier vs. rhBMP-12(0.2 mg/mL)/ACS in contralateral defects. Three animals received rhBMP-12(1.0 mg/ml)/ACS vs. rhBMP-2(0.2 mg/ml)/ACS (total implant volume/defect approximately 1 ml). The animals were euthanized 8 weeks postsurgery and block biopsies were processed for histometric analysis. RESULTS: Bone regeneration appeared increased in sites receiving rhBMP-2/ACS compared to sites receiving rhBMP-12/ACS. Cementum regeneration was similar comparing sites implanted with rhBMP-2/ACS to sites implanted with rhBMP-12/ACS. In contrast, sites receiving rhBMP-12/ACS exhibited a functionally oriented PDL bridging the gap between newly formed bone and cementum whereas this was a rare observation in sites receiving rhBMP-2/ACS. Ankylosis appeared increased in sites receiving rhBMP-2/ACS compared to those receiving rhBMP-12/ACS. CONCLUSIONS: The outcomes of this study suggest that rhBMP-12 may have significant effects on regeneration of the PDL. Additional preclinical evaluation is needed to confirm these initial observations prior to clinical application.     

Regeneration of oxytalan fibres in different types of periodontal defects: a histological study in monkeys

The aim of the present study was to investigate in monkeys the regrowth of oxytalan fibres in different types of acute and chronic periodontal defects following regenerative periodontal treatment. One-wall intrabony and mandibular furcation III-defects were produced surgically in 3 monkeys (Macaca fascicularis). After a 6-wk dental plaque accumulation period the defects were exposed using a full thickness flap procedure. The granulation tissue was removed and the root surfaces were scaled and planed. Additionally, fenestration-type defects were produced at the vestibular surfaces of the maxillary and mandibular canines by surgically removing the vestibular bone plates and the root cementum. Subsequently, the defects were treated with guided tissue regeneration (GTR), enamel matrix proteins (EMP), combination of EMP and GTR or with coronally repositioned flaps. The postoperative care included tooth cleaning once a week during the experiment. After 5 months the animals were sacrificed and and the block sections were embedded in paraffin. Eight μm histological sections were cut and stained with the oxone-aldehyde-fuchsin-Halmi method. The results revealed that in all specimens where a regenerated periodontal ligament could be observed newly formed oxytalan fibers were present. They had a mainly apico-occlusal orientation and were localized closer to the cementum than to the alveolar bone. The regenerated oxytalan fibers had a similar morphological appearance than those observed in the original periodontal ligament regardless of the treatment modality by which regeneration was accomplished. Their presence was related to that of newly formed cementum suggesting a strong relationship between these 2 tissues. The neoformation of oxytalan fibres can thus be observed in some types of periodontal defects where the cementum and the periodontal ligament have been regenerated.     

组织工程细胞片在牙周组织再生中的作用

牙周组织工程系指将在体外以理想的种子细胞和细胞外基质构建的具有生命活性的复合体植入牙周缺损部位,对牙周缺损部位进行结构功能性重建。组织工程细胞膜片构建主要有温敏培养皿、刮取法、胶原凝胶法、磁力组织工程法、表面粗糙颗粒单分子层法和聚合电解质等。牙周组织工程细胞片主要有牙周膜干细胞和骨髓间充质干细胞片,分别将其与不同的载体叠加并植入动物体内,可发现细胞外基质和纤维黏附,牙骨质样组织和牙周组织再生。将人牙周膜成纤维细胞和人脐静脉内皮细胞、人真皮成纤维细胞和人脐静脉内皮细胞共培养,所形成的细胞片可生成类似原生血管的管状结构。目前的组织工程细胞片的生物力学性能较差,与传统支架结合仍然存在着材料降解引起的组织纤维化和免疫排斥等问题,组织工程细胞片在牙周组织工程应用中具有较大应用前景;但是,要实现牙周组织的完全再生,其技术有待进一步的研究。本文就细胞片的构建、不同的牙周组织工程细胞片、细胞片工程面临的问题等研究进展作一综述。     

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.     

Cementum-periodontal ligament complex regeneration using the cell sheet technique

Background and Objective: In the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament–cementum complex. Material and methods: Human periodontal ligament cells were isolated and then cultured in dishes coated with a temperature‐responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and β‐glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed. Results: After 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral‐like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum‐like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum‐like tissue. Conclusion: This study suggests that the multilayered temperature‐responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament–cementum complex.     

Mechanical removal of necrotic periodontal ligament by either Robinson bristle brush with pumice or scalpel blade. Histomorphometric analysis and scanning electron microscopy

One of the important factors accounting for successful delayed replantation of avulsed teeth is seemingly the type of root surface treatment. Removal of necrotic cemental periodontal ligament remnants may prevent the occurrence of external root resorption, which is the major cause of loss of teeth replanted in such conditions. The purpose of this study was to compare the efficacy of two mechanical techniques for removal of root-adhered periodontal ligament. Preservation or removal of the cementum layer concomitantly with these procedures was also assessed. Forty-five roots of healthy premolars extracted for orthodontic purposes were selected. After extraction, the teeth were kept dry at room temperature for 1 h and then immersed in saline for rehydration for an additional 10 min. Thereafter, the roots were assigned to three groups, as follows: group 1 (control)--the cemental periodontal ligament was preserved; group 2--removal of the periodontal ligament by scraping root surface with a scalpel blade (SBS); group 3--periodontal ligament remnants were removed using a Robinson bristle brush at low-speed with pumice/water slurry (RBP). The specimens were analysed histomorphometrically and examined by scanning electron microscopy. The quantitative and qualitative analyses of the results showed that the RBP technique was significantly more effective than the SBS technique for removal of the periodontal ligament remnants adhered to root surface. Both techniques preserved the cementum layer.