Maximizing phenotype constraint and extracellular matrix production in primary human chondrocytes using arginine–glycine–aspartate concentration gradient hydrogels

New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine–glycine–aspartic acid peptide (RGD) concentrations from 0 mM to 10 mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.     

Bi-layer collagen/microporous electrospun nanofiber scaffold improves the osteochondral regeneration

An optimal scaffold is crucial for osteochondral regeneration. Collagen and electrospun nanofibers have been demonstrated to facilitate cartilage and bone regeneration, respectively. However, the effect of combining collagen and electrospun nanofibers on osteochondral regeneration has yet to be evaluated. Here, we report that the combination of collagen and electrospun poly-l-lactic acid nanofibers synergistically promotes osteochondral regeneration. We first fabricated bi-layer microporous scaffold with collagen and electrospun poly-l-lactic acid nanofibers (COL-nanofiber). Mesenchymal stem cells were cultured on the bi-layer scaffold and their adhesion, proliferation and differentiation were examined. Moreover, osteochondral defects were created in rabbits and implanted with COL-nanofiber scaffold. Cartilage and subchondral bone regeneration were evaluated at 6 and 12 weeks after surgery. Compared with COL scaffold, cells on COL-nanofiber scaffold exhibited more robust osteogenic differentiation, indicated by higher expression levels of OCN and runx2 genes as well as the accumulation of calcium nodules. Furthermore, implantation of COL-nanofiber scaffold seeded with cells induced more rapid subchondral bone emergence, and better cartilage formation, which led to better functional repair of osteochondral defects as manifested by histological staining, biomechanical test and micro-computed tomography data. Our study underscores the potential of using the bi-layer microporous COL-nanofiber scaffold for the treatment of deep osteochondral defects.     

基于MRI的成人正常膝关节软骨形态学测量

目的：了解正常膝关节软骨分布及股骨髁承重区的软骨厚度特点,为膝关节病变早期诊断和动态观察提供依据。方法：选取100名成人志愿者,男、女各50名,年龄20—30岁,平均25．3岁,膝关节均行MRI扫描。应用MIMICS软件对图像进行处理,在矢状位窗面上测量膝关节各部位软骨最大厚度。选择膝关节正常运动时的承重区,测量承重区域软骨的最大厚度并与其他区域进行比较。基于MRI图像行膝关节软骨三维重建,观察膝关节软骨的三维形态特征。结果：膝关节各部位的最大软骨厚度如下。胫骨外侧平台3．19mm,胫骨内侧平台3．07mm,股骨外侧髁2．93mm,股骨内侧髁3．19mm,股骨滑车软骨3．57mm,髌软骨3．75mm。不同性别与左右侧之间的软骨厚度差异无统计学意义（P〉0．05）。胫股关节承重区软骨厚度大于其他区域,有统计学差异（P〈0．05）。结论：成人正常膝关节各区域软骨厚度不同,胫股关节承重区软骨厚度大于非承重区域。基于MRI可以准确地测量膝关节各部软骨的厚度及重建膝关节软骨的三维形态。     

Pantomography of Flat Layers

The effects of in vivo zinc deficiency and restricted food intake, on the in vitro synthesis of glycosaminoglycans of rib cartilage were studied in the rat. ³⁵S-sulfate and ¹⁴C-glucosamine were used as precursors. The glycosaminoglycans were separated on microcolumns and specific radioactivities determined for the different fractions.

Chemical analyses showed that zinc deficiency or reduced food intake did not cause any qualitative or quantitative changes in the glycosaminoglycans. The radioassays indicated that zinc deficiency and reduced food intake, alone or combined, caused a somewhat lowered synthetic rate of chondroitin sulfate. In the discussion it is underlined that it seems difficult to determine conclusively the importance of zinc for the formation of the mucopolysaccharides through further in vivo deficiency studies, because of the difficulties to control and evaluate the inanition factor.     

Effect of different doses of transforming growth factor-β1 on cartilage and subchondral bone in osteoarthritic temporomandibular joints

Transforming growth factor-beta (TGF-β) plays an important part in the repair of cartilage in osteoarthritis. It has been hypothesised that intra-articular injection of TGF-β₁ promotes repair of cartilage and protects the subchondral bone from damage in osteoarthritic temporomandibular joints (TMJs). We made bilateral partial perforations of the disc to induce osteoarthritic joints in 36 rabbits. TGF-β₁ 20, 40, or 80 ng were injected into the right joint, and vehicle alone was injected into the left joint. Four additional animals were used as normal controls. Microcomputed tomography was used to quantify the three-dimensional microarchitecture of subchondral bone, followed by assessment of the proteoglycan content. All joints treated with TGF-β₁ were covered by a layer of well-organised fibrocartilage, and had increased proteoglycan content and normal microarchitectural properties, whereas the joint treated by vehicle alone had typical osteoarthritis-related degradation of cartilage and sclerosis of subchondral bone. These results suggested that TGF-β₁ is an effective way of treating osteoarthritis of the TMJ.     

Recent trends in cartilage regenerative medicine and its application to oral and maxillofacial surgery

Greater progress has been made in the clinical application of cartilage regenerative medicine, compared with that of other organs. A typical example of cartilage regenerative medicine is autologous chondrocyte implantation, in which chondrocytes isolated from the patient's cartilage are cultured and injected into the cartilage defects in a liquid- or gel-form. However, the classic autologous chondrocyte implantation has been applicable to only limited diseases, including focal cartilage lesion. Therefore, we developed “implant-type” tissue-engineered cartilage that shows mechanical strength and three-dimensional shape. This type of tissue-engineered cartilage uses scaffold composed of atelocollagen hydrogel and poly-l-lactic acid porous material, which is administered with cultured autologous auricular chondrocytes. Its clinical application to nasal deformity of cleft lip and palate patients has been ongoing at present. This review presents an overview of the current situation regarding cartilage regenerative medicine, as well as introducing our research and the development of implant-type tissue-engineered cartilage for the cleft-lip nose. The discussion of the future development of regenerative medicine is also mentioned.     

Ⅱ型胶原在T-2毒素诱导大鼠关节软骨早期损伤中的干预作用

目的 观察Ⅱ型胶原在T-2毒素诱导大鼠关节软骨早期损伤中的干预作用,在分子水平上寻找软骨损伤及修复的分子学生物标志,为探讨关节软骨损伤疾病的防治措施提供理论依据.方法 Wistar大鼠80只,按体质量随机分为4组:阴性对照组、阳性对照组、高剂量干预组、低剂量干预组,每组20只.阴性对照组食用常规成品颗粒饲料,其他3组食用含100 ng/kg T-2毒素染毒饲料;阴性对照组和阳性对照组饮自来水;低、高剂量干预组饮用含Ⅱ型胶原0.5、5.0 g/L的自来水.在3、5个月时处死大鼠,光镜下观察大鼠透明软骨的组织病理学改变,用酶联免疫吸附试验(ELISA法)检测大鼠血清Ⅱ型胶原羧基末端肽(CTX-Ⅱ)、软骨寡聚基质蛋白(COMP)及尿中吡啶啉(DPD)含量.结果 光镜下阳性对照组大鼠关节软骨细胞排列紊乱,软骨细胞变形、变性,可见大面积的软骨细胞坏死,而高、低剂量干预组表现为软骨表面原纤维形成,表层软骨细胞肿胀变圆,扁平的软骨细胞减少,软骨细胞簇集等骨关节炎早期病理改变.在3、5个月时,阴性对照组、阳性对照组、高剂量干预组、低剂量干预组大鼠血清CTX-Ⅱ含量分别为(18.77±4.61)、(25.07±9.17),(24.43±5.23)、(39.17±10.49),(21.11±5.02),(33.20±9.74),(19.87±4.53)、(29.73±9.32)μg/L;血清COMP含量分别为(5.43±2.75)、(6.38±2.23),(21.37±4.72)、(24.52±4.26),(17.27±4.77)、(20.32±4.74),(20.13±5.07)、(19.44±4.92)μg/L.其中,3个月时,与阴性对照组比较,阳性对照组血清CTX-Ⅱ含量明显升高(P＜0.05),而低、高剂量干预组未见明显改变(P均＞ 0.05);5个月时,与阴性对照组比较,其他3组血清CTX-Ⅱ含量明显升高(P均＜ 0.05),而高、低剂量干预组明显低于阳性对照组(P均＜0.05).3个月时,与阴性对照组比较,其他3组血清COMP含量明显升高(P均＜0.05),而与阳性对照组比较,高剂量干预组血清COMP含量明显降低(P＜0.05);5个月时,与阴性对照组比较,其他3组血清COMP含量明显升高(P均＜0.05),而与阳性对照组比较,高、低剂量干预组血清COMP含量明显降低(P均＜0.05).3、5个月时,上述4组大鼠尿液DPD含量分别为( 3.47±2.20)、(4.14±1.06),(4.09±2.48)、(4.33±3.43),(3.86±2.31)、(5.72±3.89),(3.58±2.77)、(4.23±2.90)μg/L,组间比较,差异无统计学意义(F值分别为2.608、2.436,P均＞0.05).结论 Ⅱ型胶原能拮抗T-2毒素的软骨损伤作用,延缓关节软骨的破坏进程,降低大鼠血清中CTX-Ⅱ及COMP水平.     

人类风湿关节炎滑膜成纤维细胞-软骨-重度联合免疫缺陷鼠模型的研制

目的 建立人类风湿关节炎(RA)滑膜成纤维细胞(SFs)-软骨-重度联合免疫缺陷(SCID)小鼠模型,以骨关节炎滑膜成纤维细胞(OASFs)作对照,探讨RASFs在RA发病中的作用及机制.方法 将培养传代至第4代的RASFs/OASFs经5-溴脱氧尿嘧啶核苷体(5-Brdu)标记后注入可吸收明胶海绵,与人的正常软骨一起移植入SCID小鼠背部皮下,第30天处死模型,剪取移植物和鼠双膝关节作组织学观察,免疫组织化学检测5-Brdu和Vimentin阳性细胞,酶联免疫吸附试验(ELISA)检测血清中人基质金属蛋白酶(MMP)-3和人白细胞介素(IL)-6含量.统计学方法采用两独立样本秩和检验.结果 ①2组血清中均可检测出人MMP-3,仅在RASFs组检测到1例IL-6.②RASFs组移植的软骨侵蚀程度(0.6±0.7与0.3±0.5)和软骨降解程度(2.3±0.8与1.7±1.0)较OASFs组有增高趋势,但差异均无统计学意义(P＞0.05).③RASFs组鼠膝关节的滑膜增生程度(3.1±0.8与1.7±1.0,P＜0.01)和软骨侵蚀程度(1.6±1.7与0.6±1.4,P＜0.05)均显著高于OASFs组.④在SCID鼠皮下、移植软骨处、鼠膝关节滑膜及骨髓中检测出5-Brdu及Vimentin阳性的SFs.结论 传代培养的RASFs在SCID鼠皮下存活并保持其侵袭性生长的特点,且能迁移至远处关节中诱发关节炎症.