丝素蛋白/左旋聚乳酸支架材料与软骨细胞体外细胞相容性研究

目的:观察静电纺丝支架材料丝素蛋白/左旋聚乳酸(SF/PLLA)的体外细胞相容性,探索其作为软骨组织工程支架材料的可行性,为进一步的体内及动物实验奠定基础。方法:将兔膝关节软骨细胞与丝素蛋白/左旋聚乳酸(SF/PLLA)支架材料复合培养,在第3、7、14天分别作HE染色和阿利新蓝+核固红染色,扫描电镜检验细胞黏着情况,MTT试验检测细胞在支架上的增殖情况。结果:细胞在支架上可以获得良好的粘附,细胞增殖良好,无细胞表型的变化。结论:丝素蛋白/左旋聚乳酸(SF/PLLA)具有良好的细胞相容性,可作为软骨组织工程支架材料。     

电纺左旋聚乳酸和左旋聚乳酸羟基磷灰石纳米纤维细胞相容性的比较研究

目的：用MTT法检测电纺左旋聚乳酸和左旋聚乳酸/羟基磷灰石PLLA和PLLA/HA纳米纤维材料,对人牙周膜细胞生长曲线的影响,评价两种材料的细胞相容性。方法：通过电纺技术制备PLLA、PLLA/HA纳米纤维材料,体外分离、培养并鉴定人牙周膜细胞,用MTT法检测人牙周膜细胞在两种材料上的生长曲线,评价两种材料的细胞相容性及其用于口腔组织工程的可能性。结果：成功分离、培养了人牙周膜细胞,通过免疫组织化学染色鉴定细胞来源于中胚层,为牙周膜细胞。电纺法制备的PLLA、PLLA/HA纳米纤维材料具有三维网状空间结构,与人牙周膜细胞共培养,MTT检测结果显示,细胞能够在材料上生长增殖,在第七天时PLLA/HA组细胞增殖好于PLLA组和对照组。结论：电纺PLLA和PLLA/HA纳米纤维材料生物相容性良好,PLLA/HA组较单纯PLLA组能够促进细胞的增殖,有望成为良好的新型口腔组织工程支架材料。     

颞下颌关节盘和髁突组织工程研究现状

采用组织工程修复颞下颌关节缺损是一种新的治疗方法,但是颞下颌关节组织工程研究还处于起步阶段,并且集中在关节盘或髁突组织再生修复上。目前的研究主要包括这两种组织结构相关的种子细胞、支架和生长因子。本文分别对颞下颌关节盘和髁突组织工程研究现状做一论述。     

细胞松弛素B对山羊颞下颌关节盘细胞及细胞骨架肌动蛋白的影响

目的:肌动蛋白作为细胞骨架的重要结构蛋白,其与细胞形态和功能有着密切关系。因此本实验通过肌动蛋白抑制剂-细胞松弛素B检测细胞骨架完整性与山羊颞下颌关节盘细胞功能变化的关系。方法:检测不同浓度的细胞松弛素B对关节盘细胞增殖及形态的影响并得出最适浓度;观察最适浓度作用后细胞凋亡、粘附及肌动蛋白形态和mRNA表达的变化。结果:2 μmol/L浓度的细胞松弛素B对关节盘细胞增殖活性具有明显抑制作用,但随培养时间延长抑制率明显降低,无促进凋亡作用;细胞粘附率明显下降;与对照组相比,处理组细胞突起结构逐渐消失,形态变圆,肌动蛋白纤维丝明显的断裂,荧光强度降低;肌动蛋白mRNA表达降低。结论:细胞松弛素B可能是通过破坏肌动蛋白的结构抑制了颞下颌关节盘细胞形态及功能。     

山羊颞下颌关节盘细胞体外培养研究

目的:研究山羊颞下颌关节盘纤维软骨细胞体外培养的生物学特性及其损伤修复的细胞学基础.方法:切取1 月龄山羊颞下颌关节盘, II型胶原酶消化获得关节盘纤维软骨细胞.逐日观察细胞的形态变化, 测定其生长曲线,甲苯胺蓝染色、I型胶原免疫组化染色鉴定.透射电镜观察细胞超微结构.结果:原代培养的关节盘纤维软骨细胞以长梭形为主, 部分多角形, 7 d即可传代.甲苯胺蓝染色阳性, I型胶原免疫组化染色胞质内可见棕黄色颗粒.超微结构显示细胞分为2 种,软骨细胞样细胞和成纤维细胞样细胞;线粒体、内质网发达.结论:体外分离培养的山羊颞下颌关节盘纤维软骨细胞具有较强的增殖能力,1～3代细胞可作为颞下颌关节盘组织工程的种子细胞.     

自组装山羊颞下颌关节盘组织工程纤维软骨模型的构建

目的 构建山羊颞下颌关节盘纤维软骨自组装模型,观察自组装组织工程纤维软骨的生物学特征,为颞下颌关节盘及其他组织工程纤维软骨的进一步研究创造条件.方法 分离、培养山羊颞下颌关节盘细胞,按每井5.5×106个接种到预制的直径5 mm×深10 mm琼脂糖井内,每天换液,培养2周,观察和检测颞下颌关节盘形态和成分的变化.结果 ...     

滑液干细胞对关节盘软骨细胞的作用研究

目的:初步探讨人颞下颌关节滑液来源的间充质干细胞条件培养基对大鼠关节盘软骨细胞的作用。方法:临床收取颞下颌关节紊乱病患者的滑液干细胞进行细胞培养,三向分化验证其细胞干性。激光共聚焦显微镜下观察获取的大鼠关节盘软骨细胞Ⅰ型胶原、Ⅱ型胶原免疫荧光表达,确认细胞属性。收集滑液干细胞培养24h的上清液制作条件培养基,设置空白对照组分别培养大鼠关节盘软骨细胞,CCK-8检测大鼠关节盘软骨细胞增殖情况,实时荧光定量PCR(RT-PCR)检测培养7天、14天大鼠关节盘软骨细胞相关基因Ⅰ、Ⅱ、Ⅹ型胶原表达变化。结果:获取的滑液干细胞具有成脂、成骨、成软骨三向分化潜能;大鼠关节盘软骨细胞表达Ⅰ型胶原、Ⅱ型胶原;CCK-8结果显示条件培养基组48h后细胞增殖速度较对照组明显加快(P<0.05);第7天、14天实时荧光定量PCR显示条件培养基组Ⅹ型胶原表达明显低于对照组。结论:人颞下颌关节滑液来源的间充质干细胞条件培养基促进大鼠关节盘软骨细胞增殖,抑制关节盘软骨细胞Ⅹ型胶原表达。     

血管内皮生长因子与颞下颌关节紊乱病

颞下颌关节细胞外基质降解、滑膜组织充血增生是颞下颌关节紊乱病重要的病理改变,血管内皮生长因子是导致其病理改变的重要因子。本文就血管内皮生长因子及其受体的相关基础研究以及与颞下颌关节盘移位、骨关节病关系的研究进展作一综述。     

组织蛋白酶D在病变颞下颌关节盘组织中的表达

组织蛋白酶Ｄ与颞下颌关节破坏性疾病有关。本研究用免疫组织化学方法 ,检测组织蛋白酶Ｄ在病变颞下颌关节盘和盘后组织中的表达 ,以探讨组织蛋白酶Ｄ与颞下颌关节紊乱病病变之间的关系。1 材料和方法 :19例颞下颌关节紊乱病患者 ,其中 3例不可复性盘前移位 ,5例盘前移位伴关节盘穿孔 ,11例骨关节病伴关节盘穿孔。男 4例 ,女 16例 ,年龄 2 4～ 6 0岁。经手术切除部分病变的颞下颌关节盘与盘后组织 ,另外 1例为正常关节盘与盘后组织作为正常对照。 2 0例标本经常规固定、脱水、石蜡包埋、切片 ,每例标本切片 4张。 1张进行常规ＨＥ染色 ,另外…     

颞下颌关节盘的结构表征与组织工程研究

以关节盘变薄、透明样变和穿孔等不可逆性病变为特征的严重的颞下颌关节功能紊乱约占整个颞下颌关节疾病的5%～10%,常因疼痛和功能障碍严重影响患者的生活质量,是目前口腔医学界面临的疑难问题之一。近年来,组织工程技术的迅速发展提示,工程化颞下颌关节盘可能为治疗严重的颞下颌关节疾病提供一个新的途径。下面就颞下颌关节盘的结构与生物力学表征、关节盘组织工程细胞源、支架与接种技术以及生物和生物力学环境与前景等问题作一综述。     

Cell type and distribution in the porcine temporomandibular joint disc.

PURPOSE: Surprisingly little is known about the cellular composition of the temporomandibular joint (TMJ) disc, which is a crucial piece of the puzzle in tissue engineering efforts. Toward this end, cell types were identified and quantified regionally in the TMJ disc. MATERIALS AND METHODS: Porcine TMJ discs were examined by histology, electron microscopy, and immunohistochemistry. Histology consisted of hematoxylin and eosin staining to identify regional variation of cell type and cell numbers. Transmission electron microscopy was used to elucidate differences in organelle content and pericellular matrix between TMJ disc cells and chondrocytes from hyaline cartilage. Immunohistochemistry was used to assess the presence of smooth and skeletal muscle character in the TMJ disc. RESULTS: The overall ratio of fibroblasts to chondrocyte-like cells in the TMJ disc was approximately 2.35 to 1, with the highest relative number of chondrocyte-like cells in the intermediate zone. Electron microscopy revealed distinct differences between TMJ disc chondrocyte-like cells and chondrocytes from hyaline cartilage with respect to organelles and the pericellular region. Immunostaining identified smooth muscle in the form of vessels, which were most prominent in the anterior band. Skeletal muscle was not observed. CONCLUSION: The cells of the TMJ disc are distinctly different from cells of hyaline cartilage, and consequently should not be referred to as chondrocytes. TMJ disc cells are comprised of heterogeneously distributed subpopulations, with fibroblasts predominating over fibrochondrocytes.     

The effects of protein-coated surfaces on passaged porcine TMJ disc cells

OBJECTIVE: Implantation of synthetic temporomandibular joint (TMJ) disc replacements aimed to alleviate pain and restore functional losses caused by TMJ disorders. Unfortunately, these synthetic replacements have been largely unsuccessful and in some instances have incited severe immune responses. Tissue engineering, however, may provide viable TMJ disc replacements. Towards this end, we have studied TMJ disc gene expression as a measure of protein production potential. With passage, collagen type I and aggrecan gene expression decrease in TMJ disc cell cultures. We hypothesize that surfaces coated with TMJ disc proteins may rapidly recover the lost gene expression in passaged TMJ disc cells. DESIGN: To study these effects, passages 0, 1, and 2 TMJ disc cells were plated in wells coated with aggrecan, collagen type I, collagen type II, or decorin. Safranin O staining was conducted to visualize cell aggregation. RESULTS: At passage 0, cultures appeared similar on each surface; however, by passages 1 and 2, aggrecan-coated and decorin-coated surfaces appeared to have more cell aggregates. Gene expression data did not correspond to these visual changes. No treated surface offered a significant change in aggrecan, collagen type I, or decorin expression relative to untreated controls. Furthermore, aggrecan and collagen type I gene expression dropped relative to samples taken prior to plating. CONCLUSIONS: These results indicate that, despite visual changes described by cell aggregates, protein coatings have limited effects for recovering TMJ disc gene expression in monolayer cultures.     

Lubricin immunohistochemical expression in human temporomandibular joint disc with internal derangement

J Oral Pathol Med (2011) 40 : 587–592 Lubricin is a chondroprotective, mucinous glycoprotein which contribute to joint lubrication, especially to boundary lubrication and maintains joint integrity. The present investigation aimed to study the immunolocalization of lubricin in TMJ discs from patients affected by anterior disc displacement with reduction (ADDwR) ADDwoR. Eighteen TMJ displaced disc affected by ADDwoR were processed immunohistochemically, with a polyclonal anti‐lubricin antibody, used at 1:50 working dilution. The percentage of lubricin immunopositive cells (extent score = ES) and the extent of lubricin staining of the disc extracellular matrix (ECM), were evaluated. Each sample was scored for histopathological changes. Percentage of immunostained surface disc cells was the same (ES = 4) in both control and ADDwOR cells, being this data not statistically significant (P < 0.05). In pathological specimens the percentages of lubricin‐stained cells was very high with an ES of 4 respect to control specimen, and this difference was statistically significant different (P > 0.05). The extracellular matrix (ECM) of discs at the disc surfaces of both pathological and normal specimens was very heavily stained (++++). Both the ES and ECM staining were not statistically correlated to the TMJ degeneration score according to the Spearman’s rank correlation coefficient. According to our findings, a longstanding TMJ disc injury, affects lubricin expression in the TMJ disc tissue and not its surfaces, moreover, lubricin immunostaining is not correlated to TMJ disc histopathological changes.     

ADAMTS‐4 and ADAMTS‐5 expression in human temporomandibular joint discs with internal derangement,correlates with degeneration

Temporomandibular joint (TMJ) internal derangement (ID) is one of the most common form of temporomandibular disorders. There is evidence showing the increased expression of matrix metalloproteinases (MMPs) in the cells from degenerated TMJ disc. ADAMTS are a large family of metalloproteases which are responsible for proteoglycans degradation. The present study aimed to evaluate ADAMTS‐4 and ADAMTS‐5 immunohistochemical expression in human TMJ discs from patients affected by ID, and to find out if there is any correlation with the degree of histopathological changes. Eighteen temporomandibular displaced disc specimens and sixteen TMJ disc control were used for the present study. Specimens were immunohistochemically processed and ADAMTS‐4 and ADAMTS‐5 expression were obtained respectively for the anterior (AB), intermediate (IB) and posterior (PB) bands and compared to the histopathological degeneration score (HDS). Immunoreactivity for ADAMTS‐4 and ‐5, was observed in both not degenerated and degenerated human TMJ discs. Both the percentage of ADAMTS‐4 and ‐5 immunostained cells (ES) and the intensity of staining (IS) were significantly greater in affected specimens compared with those in control discs. The ADAMTS‐5 ES and IS of the 3 bands of the disc correlated to the TMJ disc HDS (0.001 < P < 0.05), on the other hand only AB and IB, ADAMTS‐4 immunostaining scores correlated to HDS. According to these findings it can be assumed in that the more histopathological changes in the disc are detected, the higher levels of ADAMTS are produced. This in turn can lead to ECM breakdown and in turn to a more advanced disc displacement.     

Towards resorbable 3D-printed scaffolds for craniofacial bone regeneration

This review will briefly examine the development of 3D-printed scaffolds for craniofacial bone regeneration. We will, in particular, highlight our work using Poly(L-lactic acid) (PLLA) and collagen-based bio-inks. This paper is a narrative review of the materials used for scaffold fabrication by 3D printing. We have also reviewed two types of scaffolds that we designed and fabricated. Poly(L-lactic acid) (PLLA) scaffolds were printed using fused deposition modelling technology. Collagen-based scaffolds were printed using a bioprinting technique. These scaffolds were tested for their physical properties and biocompatibility. Work in the emerging field of 3D-printed scaffolds for bone repair is briefly reviewed. Our work provides an example of PLLA scaffolds that were successfully 3D-printed with optimal porosity, pore size and fibre thickness. The compressive modulus was similar to, or better than, the trabecular bone of the mandible. PLLA scaffolds generated an electric potential upon cyclic/repeated loading. The crystallinity was reduced during the 3D printing. The hydrolytic degradation was relatively slow. Osteoblast-like cells did not attach to uncoated scaffolds but attached well and proliferated after coating the scaffold with fibrinogen. Collagen-based bio-ink scaffolds were also printed successfully. Osteoclast-like cells adhered, differentiated, and survived well on the scaffold. Efforts are underway to identify means to improve the structural stability of the collagen-based scaffolds, perhaps through mineralization by the polymer-induced liquid precursor process. 3D-printing technology is promising for constructing next-generation bone regeneration scaffolds. We describe our efforts to test PLLA and collagen scaffolds produced by 3D printing. The 3D-printed PLLA scaffolds showed promising properties akin to natural bone. Collagen scaffolds need further work to improve structural integrity. Ideally, such biological scaffolds will be mineralized to produce true bone biomimetics. These scaffolds warrant further investigation for bone regeneration.     

Role of interleukin-1 in induction of matrix metalloproteinases synthesized by rat temporomandibular joint chondrocytes and disc cells

To identify cartilage-degrading enzymes and cell types that can be specifically induced by interleukin-1 (IL-1)alpha, we studied matrix metalloproteinase (MMP) activities of cultured rat temporomandibular joint (TMJ) chondrocytes and disc cells. The cells were isolated from TMJs pre-injected with normal physiological saline (CR) or recombinant human IL-1alpha (AR). MMP activities in the conditioned media were assayed by gelatin enzymography, and they were identified by Western blot analyses. MMP mRNAs in these cells were also detected by RT-PCR. IL-1alpha significantly induced an increase of active MMP9 as well as pro- and active MMP3, but had no effect on the MMP2 activity in both types of cells. MMP3 and MMP9 mRNAs were also inducible in these cells by IL-1alpha stimulation. Furthermore, disc cells were more susceptible to IL-1alpha than chondrocytes. AR cells spontaneously produced the same MMPs in vitro as the CR cells synthesized under IL-1alpha stimulation. The results indicate that MMP9 and MMP3 were predominantly produced by disc cells, and these may be considered to play a pivotal role in ECM degradation during pathological conditions of the TMJ, such as IL-1-induced TMJ arthritis.     

The attachments of the temporomandibular joint disc: a biochemical and histological investigation

ObjectiveThe complex movement of the temporomandibular joint (TMJ) disc during mastication is controlled in large part by the disc's attachments to the surrounding tissues. This study seeks to address the lack of available quantitative data characterizing the extracellular matrix composition of the discal attachments and how these properties compare to the disc.DesignPorcine TMJ disc–attachment complexes were carefully dissected into six discal attachments and five TMJ disc regions. All samples were assayed biochemically for total collagen, glycosaminoglycan (GAG), DNA, and hydration. Additionally, histology was performed on the whole joint to investigate the anatomy of the disc–attachment complex, and to verify the regional distribution of matrix components.ResultsQuantitative biochemical assays showed that overall water content was fairly constant in all disc and attachment regions. Disc regions generally showed higher sulfated GAG and collagen content than the attachments. In contrast, the attachments contained greater DNA content than the disc. Histological staining supported the quantitative results and also indicated more elastic fibres to be present in the attachments than the disc.ConclusionsAlthough macroscopically the TMJ disc and its attachments form a seamless complex within the joint, a closer look at regional biochemical constituents reveals that these two components are distinct. Whilst the disc and attachments both contain the same major constituents, the relative amounts of these components vary based on the functional requirements of the tissue. These results can further understanding of both TMJ biology and pathology.