Effect of blood on the morphological,biochemical and biomechanical properties of engineered cartilage

The use of autologous chondrocytes seeded onto a biological scaffold represents a current valid tool for cartilage repair. However, the effect of the contact of blood to the engineered construct is unknown. The aim of this work was to investigate in vitro the effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage. Articular chondrocytes were enzymatically isolated from swine joints, expanded in monolayer culture and seeded onto collagen membranes for 2 weeks. Then, the seeded membranes were placed for 3 days in contact with peripheral blood, which was obtained from animals of the same species and diluted with a standard medium. As controls, some samples were left in the standard medium. After the 3 days’ contact, some samples were retrieved for analysis; others were returned to standard culture conditions for 21 additional days, in order to investigate the “long-term effect” of the blood contact. Upon retrieval, all seeded samples showed increasing sizes and weights over time. However, the samples exposed to blood presented lower values with respect to the controls. Biochemical evaluation demonstrated a reduction in the mitochondrial activity due to blood contact at the early culture time (3 days post blood contact), followed by a partial recovery at the longer culture time (21 days post blood contact). Histological evaluation demonstrated evident cartilage-like matrix production for both groups. Biomechanical data showed a reduction of the values, followed by stabilization, regardless of the presence of blood. Based on the data obtained in this study, we can conclude that blood contact affects the chondrocyte activity and determines a delay in the dimensional growth of the engineered cartilage; however, at the experimental times utilized in this study, this delay did not affect the histological pattern and the biomechanical properties of the construct.     

Cell-based therapy for meniscal repair: a large animal study

BACKGROUND: The avascular portion of the meniscus cartilage in the knee does not have the ability to repair spontaneously. HYPOTHESIS: Cell-based therapy is able to repair a lesion in the swine meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Sixteen Yorkshire pigs were divided into four groups. A longitudinal tear was produced in the avascular portion of the left medial meniscus of 4 pigs. Autologous chondrocytes were seeded onto devitalized allogenic meniscal slices and were secured inside the lesion with two sutures. Identical incisions were created in 12 other pigs, which were used as three separate control groups: 4 animals treated with an unseeded scaffold, 4 were simply sutured, and 4 were left untreated. Meniscal samples were collected after 9 weeks, and the samples were analyzed grossly, histologically, and histomorphometrically. RESULTS: Gross results showed bonding of the lesion margins in the specimens of the experimental group, whereas no repair was noted in any of the control group specimens. Histological and histomorphometrical analysis showed multiple areas of healing in the specimens of the experimental group. CONCLUSIONS: This study demonstrated the ability of seeded chondrocytes to heal a meniscal tear. Clinical Relevance: Cell-based therapy could be a potential tool for avascular meniscus repair.     

Autologous collagen-induced chondrogenesis technique (ACIC) for the treatment of chondral lesions of the talus

Purpose

Autologous collagen-induced chondrogenesis technique (ACIC) combines microfractures with the use of an injectable atelocollagen matrix that allows performing the whole cartilage repair treatment arthroscopically. The aim of this study was to evaluate the in vitro cytocompatibility of this biomaterial using human bone marrow mesenchymal stem cells and human chondrocytes. Moreover, the preliminary data of five patients affected by chondral lesion of the talus treated with the ACIC technique are shown.

Methods

Human bone marrow mesenchymal stem cells and human chondrocytes were seeded on solid and pre-solid atelocollagen scaffolds. Cell–scaffold constructs were cultured for 7 days and then prepared for histological analyses. Arthroscopic ACIC was performed in five patients affected by chondral lesions of the talus; they were clinically evaluated with AOFAS, VAS and Tegner score before and then after 6 months from surgery.

Results

In vitro results showed that both bone marrow mesenchymal stem cells and chondrocytes were able to efficiently colonize the whole construct, from the surface to the core, only when seeded on the pre-solid atelocollagen scaffold, but not on its solid form. No adverse events were observed in the patients treated with the ACIC technique; a significant improvement in VAS pain scale and in AOFAS score was found at 6 months follow up.

Conclusion

Injectable atelocollagen can be considered a feasible scaffold for cartilage repair treatment, in particular if used in its pre-solid form. ACIC leads to good clinical results in the treatment for chondral lesions of the talus even if longer follow-up and a higher number of patients are necessary to confirm these data.

Level of evidence

IV.     

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies

Purpose

To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide^®) scaffold.

Methods

By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water–dioxane–PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT–PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide^® scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O’Driscoll score.

Results

In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide^® scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide^® scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells.

Conclusion

The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide^® scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide^® scaffold with cells.     

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

The effect of microRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel

The objective of this study was to investigate the effect of microRNA-21 (miR-21) on the proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Articular cartilage was harvested aseptically from the knee and hip joints of rats. In the experimental group, double-stranded miR-21 was transfected into the chondrocytes, and in the control group, scrambled siRNA was used. After that, chondrocytes were cultured in atelocollagen gel for 3 weeks. At 1, 2, and 3 weeks after transfection, the cell numbers were counted, and the expression levels of Col2a1 and aggrecan were measured by real-time PCR. Histological analysis by toluidine blue staining was performed at 3 weeks. The cell number in the experimental group rapidly increased compared to the control group. The expression levels of Col2a1 and aggrecan in the experimental group were higher than in the control. Histological analysis revealed that many more cells with a metachromatic stain were present in the experimental group than in the control. This study demonstrated that miR-21 promotes high proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel.     

不同细胞移植修复兔全层关节软骨缺损的比较研究

目的 :比较软骨细胞、骨髓基质细胞及成纤维细胞对全层关节软骨缺损的修复作用。材料和方法 :取幼兔的软骨细胞、骨髓基质细胞及成纤维细胞 ,共 3种有生成软骨潜力的细胞进行体外分离培养 ;以聚乳酸 (PLA)为载体 ,将培养的原代细胞植入PLA支架上 ,形成细胞 -PLA复合物。于 2 8只成年新西兰大白兔的股骨滑车关节面上造成直径 4 5mm、深 3 0mm的全层关节软骨缺损 ,将 3种细胞 -PLA复合物分别植入关节软骨缺损处。植入细胞 -PLA复合物为实验组 ,单纯植入PLA支架为对照组。术后 6周、12周观察缺损修复情况及新生组织类型。结果 :软骨细胞移植组为软骨样组织修复 ,分界明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨部分重建 ;细胞排列紊乱。骨髓基质细胞移植组为软骨样组织修复 ,分界不明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨重建良好 ,软骨下潮线恢复 ;细胞排列趋于正常。成纤维细胞移植组为纤维组织修复 ,甲苯胺兰及Ⅱ型胶原染色阴性 ;软骨下潮线消失。对照组为纤维组织修复。结论 :软骨细胞、骨髓基质细胞移植修复软骨缺损明显优于成纤维细胞及对照组。骨髓基质细胞与软骨细胞移植组的修复结果无统计学差异 ,但骨髓基质细胞修复组织的细胞排列有序 ,软骨下骨重建良好 ,与周围组织融合密切 ,更接近正?     

同种异体软骨细胞/聚乳酸复合物即时修复兔耳廓软骨缺损

 目的观察用同种异体软骨细胞/聚乳酸(Poly-DL-lactide,PDLLA)复合物在体内即时修复软骨缺损的能力.方法将兔耳廓软骨细胞体外分离消化,以PDLLA为支架,用软骨细胞/PDLLA复合物即时移植修复兔耳廓软骨缺损,对照组采用PDLLA,6、12、18周后观察软骨缺损修复情况.结果实验组移植后18周,软骨缺损愈合,修复软骨厚度均匀.对照组缺损区为条索状纤维组织修复,中央部凹陷.结论同种异体软骨细胞/PDLLA复合物即时修复软骨缺损是一种非常有前途的且适合临床应用的组织工程学方法.     

Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?: a morphological and molecular study of OCD fragments

Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in “bundles” and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae close to mineralized “islands” in the cartilage and hypertrophic chondrocytes containing accumulated matrix proteins. Immunostaining revealed: (1) that types I, II, VI and X collagens and aggrecans were deposited intracellulary and (2) co‐localization within the islands of types I, II, X collagens and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non‐conclusive, only single nucleotide polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification.     

脐带全层源间充质干细胞分离培养及向成软骨细胞分化的实验研究

目的从人脐带全层中分离培养间充质干细胞(MSCs),并进行成软骨诱导分化,为组织工程软骨和软骨损伤后修复提供种子细胞。方法采用胶原酶消化法从脐带全层中分离培养间充质干细胞,显微镜下观察细胞形态,细胞计数法绘制细胞生长曲线,采用流式细胞仪检测细胞周期及细胞表型,采用微团细胞培养在软骨诱导液中向软骨细胞分化,阿尔辛蓝及甲苯胺蓝染色检测细胞分化情况,RT-PCR法检测诱导后细胞表达聚集蛋白聚糖(ACAN)基因情况。结果人脐带全层来源的MSCs呈成纤维样形态漩涡状贴壁生长,细胞高表达HLA-I类分子、CD73、CD90、CD166及CD105,不表达CD34、CD45、CD14、CD31、CD80、CD86及HLA-DR。细胞诱导分化21d后,阿尔辛兰及甲苯胺蓝染色阳性;RT-PCR检测诱导后的细胞表达ACAN,而对照组无表达。结论人脐带全层为成体MSCs提供一种新而方便的来源,人脐带间充质干细胞(hucMSCs)体外培养能够向软骨细胞分化。     

癌基因与正常及骨关节炎中软骨细胞凋亡关系的研究

用免疫组化及ＤＮＡ缺口末端标记法观察了正常及骨关节炎软骨组织中ｃ－ｍｙｃ，ｂｃｌ－２的表达以及软骨细胞的凋亡情况。发现在正常软骨组织的表层有软骨细胞的凋亡及＆ｕ，ｃ－ｍｙｃ的表达，在移行层有ｂｃｌ－２的表达，在柱状层下层有ｃ、ｎａｙｃ的表达及ＤＮＡ缺口末端标记的阳性信号；在骨关节炎的软骨组织中发现软骨细胞凋亡及ｆａｓ表达的减少或消失，ｂｃｌ－２的表达增强。结果表明：在骨关节炎软骨组织中软骨细胞凋亡的减少可能是软骨细胞对软骨组织损伤的反应，这种反应可以延长软骨细胞的存活时间，增强软骨细胞的修复能力。     

应用生物反应器体外培养骨软骨复合体修复犬关节软骨损伤

目的 探讨应用灌注型生物反应器体外培养骨软骨复合体修复关节软骨损伤的可行性. 方法 体外分离犬骨髓间充质干细胞(MSCs),流式细胞仪鉴定.经过生长因子诱导生成软骨细胞和成骨细胞后,免疫组化及碱性磷酸酶等染色鉴定.将软骨细胞、成骨细胞接种到三维多孔β-磷酸三钙(β-TCP)陶瓷支架材料上,置于灌注型生物反应器中复合培养21 d,构建骨软骨复合体,扫描电镜观察细胞在支架材料上的黏附、伸展和增殖情况,并模仿马赛克骨软骨移植术用塑形良好的骨软骨复合体修复犬软骨缺损,切片染色观察其修复情况. 结果 扫描电镜显示软骨细胞、成骨细胞在β-TCP支架上的黏附、伸展和增殖良好,实验组缺损区软骨厚度与正常软骨组织接近.实验组与阴性时照组比较,差异有统计学意义(q=12.337 0,P＜0.01);与空白对照组比较,差异有统计学意义(q=31.5393,P＜0.01). 结论 灌注型生物反应器使软骨和成骨细胞在三维载体内存活并增殖,提高细胞在载体内的复合效率.     

An allogenic cell-based implant for meniscal lesions

BACKGROUND: Meniscal tears in the avascular zones do not heal. Although tissue-engineering approaches using cells seeded onto scaffolds could expand the indication for meniscal repair, harvesting autologous cells could cause additional trauma to the patient. Allogenic cells, however, could provide an unlimited amount of cells. HYPOTHESIS: Allogenic cells from 2 anatomical sources can repair lesions in the avascular region of the meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Both autologous and allogenic chondrocytes were seeded onto a Vicryl mesh scaffold and sutured into a bucket-handle lesion created in the medial menisci of 17 swine. Controls consisted of 3 swine knees treated with unseeded implants and controls from a previous experiment in which 4 swine were treated with suture only and 4 with no treatment. Menisci were harvested after 12 weeks and evaluated histologically for new tissue and percentage of interface healing surface; they were also evaluated statistically. RESULTS: The lesions were closed in 15 of 17 menisci. None of the control samples demonstrated healing. Histologic analysis of sequential cuts through the lesion showed formation of new scar-like tissue in all experimental samples. One of 8 menisci was completely healed in the allogenic group and 2 of 9 in the autologous group; the remaining samples were partially healed in both groups. No statistically significant differences in the percentage of healing were observed between the autologous and allogenic cell-based implants. CONCLUSION: Use of autologous and allogenic chondrocytes delivered via a biodegradable mesh enhanced healing of avascular meniscal lesions. CLINICAL RELEVANCE: This study demonstrates the potential of a tissue-engineered cellular repair of the meniscus using autologous and allogenic chondrocytes.     

Ionizing radiation causes active degradation and reduces matrix synthesis in articular cartilage

Abstract

Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1.

Materials and methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours.

Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy.

Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.     

Patellofemoral full-thickness chondral defects treated with Hyalograft-C: a clinical, arthroscopic, and histologic review

BACKGROUND: Tissue engineering has emerged as a potential therapeutic option for cartilage regeneration. HYPOTHESIS: Hyaluronan-based scaffolds seeded with autologous chondrocytes are a viable treatment for damaged articular surface of the patellofemoral joint. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Thirty-two chondral lesions with a mean size of 4.7 cm(2) were treated with Hyalograft-C. Twenty-two lesions were located in the patella and 10 in the trochlea. Sixteen patients had previous trauma, 3 had osteochondritis dissecans, and 13 had degenerative changes. Transplantations were carried out arthroscopically or through a miniarthrotomy incision. Eight patients had concomitant procedures, including patellar realignment (2), lateral release (3), and meniscectomy (3). Results were evaluated using the International Cartilage Repair Society-International Knee Documentation Committee scale, EuroQol EQ-5D form, and magnetic resonance imaging scans at 12 and 24 months. Six patients had second-look arthroscopy and biopsies. Statistical analysis was performed using the paired t test and Wilcoxon signed rank test. RESULTS: The International Cartilage Repair Society-International Knee Documentation Committee and EuroQol EQ-5D scores demonstrated a statistically significant improvement (P < .0001). Objective preoperative data improved from 6/32 (18.8%) with International Knee Documentation Committee A or B to 29/32 (90.7%) at 24 months after transplantation. Mean subjective scores improved from 43.2 points preoperatively to 73.6 points 24 months after implantation. Magnetic resonance imaging studies at 24 months revealed 71% to have an almost normal cartilage with positive correlation to clinical outcomes. Second-look arthroscopies in 6 cases revealed the repaired surface to be nearly normal with biopsy samples characterized as hyaline-like in appearance. CONCLUSION: Biodegradable scaffolds seeded with autologous chondrocytes can be a viable treatment for chondral lesions. The type of tissue repair achieved demonstrated histologic characteristics similar to normal articular cartilage. Long-term investigations are needed to determine the durability of the repair produced with this technique.     

In vitro uptake 153gadolinium and gadolinium complexes by hyaline articular cartilage.

This in vitro study evaluated whether Gadolinium (Gd) penetrates into hyaline cartilage and would be incorporated into vital chondrocytes. Hyaline joint cartilage of rabbits was exposed to radioactive 153GdCl3 and to a radioactive 153Gd-DTPA-BSA-complex (DTPA, diethylene-triaminepentaacetic acid; BSA, bovine serum albumine). In addition an exchange experiment with radioactive 153GdCl3 versus Gd-DTPA-di-N-methylglucamine (Magnevist) was performed. Incorporation of 153GdCl3 into neuroblastoma cells, connective tissue cells and chondrocytes was tested. The results showed that the depth and extent of incorporation of Gd depends on the molecular mass and time of exposure. 153Gd-DTPA-BSA complexes exhibited an incorporation rate of maximal 11% +/- 2.8% up to the middle third of the cartilage within 24 h with almost no incorporation (2 +/- 1.9%) for the deep layer. The exchange experiment revealed no uptake of Gd for the deep layer. The maximal incorporation rate of 153GdCl3 into vital chondrocytes was 6.3%. These data indicate that under the condition of MR-arthrography, Gd-DTPA-di-N-methylglucamine will not be absorbed into the deep layers of hyaline cartilage and will not be incorporated into vital chondrocytes.     

聚乙醇酸负载同种异体软骨细胞移植修复兔关节软骨缺损

目的：应用聚乙醇酸（ＰＧＡ）负载的兔软骨细胞培养移植修复同种异体关节软骨缺损．方法：应用在生物体内可降解吸收、纤维状多孔态的ＰＧＡ作为支架行兔软骨细胞培养．培养１４天后，软骨细胞在ＰＧＡ提供的三维空间中大量分裂、增殖并合成大量软骨基质，形成ＰＧＡ－软骨细胞复合体，然后利用该复合体移植修复同种异体兔膝关节全层软骨缺损，对侧膝关节作对照．术后行大体、组织学、电镜动态观察及修复组织厚度测定．结果：ＰＧＡ在术后８周完全降解吸收，实验侧与对照侧修复组织的厚度有显著性差异（Ｐ＜０．０１）；术后１６周在实验侧可见典型的软骨组织，电镜下为成熟的软骨细胞，而对照侧为纤维组织修复．结论：应用ＰＧＡ－软骨细胞复合体移植，可修复同种异体的兔关节软骨缺损，为临床治疗关节软骨缺损奠定了基础．     

诱导型一氧化氮合酶和白介素1受体拮抗剂在骨关节炎滑膜和软骨中的表达及意义

炎症前细胞因子白介素 1(IL - 1)通过刺激软骨细胞合成诱导型一氧化氮合酶 (iNOS) ,产生一氧化氮(NO) ,增加基质降解 ,参与关节炎的炎症活动 ,而局部产生的白介素 1受体拮抗剂蛋白 (IL - 1Ra)可阻止软骨破坏。本实验旨在观察iNOS和IL - 1Ra在骨关节炎 (OA)关节滑膜和软骨中的表达及分布。我们收集了 6例OA患者的关节滑膜和软骨标本。通过免疫组化方法对iNOS、CD6 8和IL - 1Ra进行了原位检测。在 6例OA滑膜标本连续切片中 ,滑膜的衬里层细胞、炎症浸润细胞以及血管内皮细胞中均强烈表达iNOS蛋白。IL - 1Ra阳性细胞主要局限于滑膜的衬里层细胞及血管内皮细胞 ,表达强度低于iNOS表达。CD6 8阳性细胞 (检测巨噬细胞的抗体 )仅在滑膜衬里层的表层 (A型滑膜细胞 )及炎症浸润细胞中零星表达。在 6例骨关节炎软骨连续切片标本中 ,iNOS和IL - 1Ra在软骨全层及软骨下骨髓腔内均有强烈表达。结果提示 ,骨关节炎时 ,iNOS主要产生于滑膜衬里层细胞、软骨细胞以及血管内皮细胞 ,而IL - 1Ra主要产生于滑膜衬里层的巨噬样滑膜细胞和软骨细胞 ;iNOS和IL - 1Ra之间的拮抗作用可能在骨关节炎的病理机制中发挥很重要的作用。     

半月板纤维软骨细胞与壳聚糖/聚磷酸钙体外复合培养的实验研究

目的:观察半月板纤维软骨细胞在不同配比壳聚糖/聚磷酸钙(chitosan/calciumpolyphosphate,CS/CPP)支架材料上的生长状况,优选最佳配比CS/CPP生物材料作为组织工程半月板支架材料。方法:采用机械分离与酶连续消化相结合的方法体外分离兔半月板纤维软骨细胞,单层培养传代至第3代,并对其进行表型鉴定。采用共混-化学交联固化-冷冻干燥法将CS、醛基化海藻酸钠(aldehyde alginate,ADA)、CPP有机地结合起来,制备4种不同配比的新型组织工程半月板复合支架材料。将体外分离培养的第3代半月板细胞,通过二次沉淀接种法将其种植于不同配比的CS/CPP支架上,体外培养7天,采用相差倒置显微镜、SEM、HE染色观察细胞在支架上的形态、黏附、生长情况。结果:体外分离培养的第3代半月板细胞基本维持纤维软骨细胞的表型。相差倒置显微镜下可见,细胞在支架上黏附良好;扫描电镜下可见,细胞在支架上均匀分布,细胞多数呈多角形,并有基质分泌;HE染色结果显示,有细胞长入到三维支架材料内部。其中,半月板细胞在3:7的CS/CPP支架材料上单位面积内数目最多,生长最旺盛,细胞外基质分泌最多。结论:三维多孔的CS/CPP复合材料能促进半月板纤维软骨细胞的黏附、生长和增殖,其中3:7的CS/CPP支架材料细胞相容性和生物活性最好,最适于半月板纤维软骨细胞粘附和生长,并且能促进半月板细胞增殖和维持其表型,有望成为组织工程半月板良好的支架载体。     

Therapieoptionen zur Behandlung von Knorpelschäden: Techniken und Indikationen

BACKGROUND: Cartilage is composed of chondrocytes embedded within an extracellular matrix of collagens, proteoglycans, and noncollagenous proteins. Together, these structures maintain the unique mechanical properties and manifest its striking inability to heal even the most minor injury. METHODS: This review presents the principles of cartilage structure and the biological background of cartilage repair and gives information about the surgical techniques for treating cartilage defects. RESULTS: The response of cartilage to injuries differs from that of other tissues because of its avascularity, the immobility of chondrocytes and the limited ability of mature chondrocytes to proliferate and alter their synthetic patterns. Surgical therapeutic efforts in treating cartilage defects have focused on bringing new cells and tissues capable of chondrogenesis into the lesions and facilitating the access to the vascular system. CONCLUSION: The right indication and the treatment of joint instability and axis deformation are essential for the successful use of cartilage repair procedures.