A tissue engineered osteochondral plug: an in vitro morphological evaluation

Articular cartilage lesions have a poor intrinsic healing potential. The repair tissue is often fibrous, having insufficient biomechanical properties, which could frequently lead to the development of early osteoarthritis. In the last decade, tissue engineering approaches addressed this topic in order to restore joint function with a differentiated and functional tissue. Many biomaterials and techniques have been proposed and some of them applied in clinical practice, even though several concerns have been raised on the quality of the engineered tissue and on its integration in the host joint. In this study, we focused on engineering in vitro a biphasic composite made of cellular fibrin glue and a calcium–phosphate scaffold. Biphasic composites are the latest products of tissue engineering applied to articular cartilage and they seem to allow a more efficient integration of the engineered tissue with the host. However, a firm in vitro bonding between the two components of the composite is a necessary condition to validate this model. Our study demonstrated a gross and microscopic integration of the two components and a cartilage-like quality of the newly formed matrix. Moreover, we noticed an improvement of this integration and GAGs production during the in vitro culture.     

An in vitro tissue-engineered model for osteochondral repair

Abstract One of the main topics of regenerative medicine and tissue engineering is to address the problem of lesions involving articular cartilage. In fact, these lesions do not heal spontaneously and often lead to osteoarthritis, which causes chronic pain and worsens quality of life. Moreover, the only available treatment for osteoarthritis is symptomatic therapy and prosthetic replacement, with far from satisfactory results. A more conservative approach that restores the articular surface and function with a biologic tissue is desirable. Several strategies for regenerating articular cartilage have been proposed and applied in clinical practice but a gold standard has not yet been identified. Biphasic composites are the latest products of tissue engineering applied to articular cartilage and they seem to permit a more efficient integration of the engineered neo-tissue with the host. We present an in vitro tissue engineered model for osteochondral repair based on a composite of chondrocytes-fibrin glue gel and a calciumphosphate scaffold. This composite showed a gross integration of the two components and a cartilage-like quality of the newly formed matrix. Further studies are planned to quantify the adherence between the scaffold and the cellular fibrin glue.     

Cartilage repair with chondrocytes in fibrin hydrogel and MPEG polylactide scaffold: an in vivo study in goats

Polylactic acid polymers have been used extensively as biomaterials and have shown promising properties for cartilage tissue engineering. Numerous scaffold materials exist and the optimal scaffold needs to be identified. We have tried to assess the possibilities for cartilage repair by the use of two different scaffold techniques; autologous chondrocytes in a fibrin hydrogel and a novel MPEG-PLGA scaffold, where autologous chondrocytes are immobilized within the MPEG-PLGA scaffold by a fibrin hydrogel. Twenty adult goats were used for the study. A 6 mm circular full-thickness cartilage defect was created in both medial femoral condyles. The defects were randomized to the following four treatment groups. (1) Empty defect (control). (2) Subchondral drilling (control). (3) Fibrin hydrogel with autologous chondrocytes. (4) Fibrin hydrogel/chondrocyte solution in a MPEG-PLGA porous scaffold. Animals were followed for 4 month. Eight defects in each treatment group completed the study. ICRS macroscopic scoring (0-12). Indentation test was performed to assess stiffness of repair tissue. Histological analyses was performed using O'Driscoll and Pineda cartilage scores as well as percentage tissue filling of the defects. The MPEG-PLGA/chondrocytes scaffold was the superior treatment modality based on the macroscopic surface score, histological scores and defect filling. The mechanical test demonstrated no difference between treatment groups. The MPEG-PLGA/chondrocyte composite demonstrated significantly better cartilage repair response than empty defects, osteochondral drilling and fibrin hydrogel with chondrocytes. The novel MPEG-PLGA scaffold in combination with chondrocytes need further studies with respect to longer follow-up times.     

Early postoperative adherence of matrix-induced autologous chondrocyte implantation for the treatment of full-thickness cartilage defects of the femoral condyle

Matrix-induced autologous chondrocyte implantation (MACI) is a tissue-engineering technique for the treatment of full-thickness articular cartilage defects and requires the use of a three-dimensional collagen type I–III membrane seeded with cultured autologous chondrocytes. The cell-scaffold construct is implanted in the debrided cartilage defect and fixed only with fibrin glue, with no periosteal cover or further surgical fixation. In a clinical pilot study, the MACI technique was used for the treatment of full-thickness, weight-bearing chondral defects of the femoral condyle in 16 patients. All patients were followed prospectively and the early postoperative attachment rate, 34.7 days (range: 22–47) after the scaffold implantation, was determined. With the use of high-resolution magnetic resonance imaging (MRI), the transplant was graded as completely attached, partially attached, or detached. In 14 of 16 patients (87.5%), a completely-attached graft was found, and the cartilage defect site was totally covered by the implanted scaffold and repair tissue. In one patient (6.25%), a partial attachment occurred with partial filling of the chondral defect. A complete detachment of the graft was found in one patient (6.25%), which resulted in an empty defect site with exposure of the subchondral bone. Interobserver variability for the MRI grading of the transplants showed substantial agreement (=0.775) and perfect agreement (_w=0.99). In conclusion, the implantation and fixation of a cell-scaffold construct in a deep cartilage defect of the femoral condyle with fibrin glue and with no further surgical fixation leads to a high attachment rate 34.7 days after the implantation, as determined with high resolution MRI.     

Replacement of the knee meniscus by a porous polymer implant: a study in dogs

BACKGROUND: Meniscectomy will lead to articular cartilage degeneration in the long term. Therefore, the authors developed an implant to replace the native meniscus. HYPOTHESIS: The porous polymer meniscus implant develops into a neomeniscus and protects the cartilage from degeneration. STUDY DESIGN: Controlled laboratory study. METHODS: In a dog model, a porous polymer scaffold with optimal properties for tissue infiltration and regeneration of a neomeniscus was implanted and compared with total meniscectomy. The tissue infiltration and redifferentiation in the scaffold, the stiffness of the scaffold, and the articular cartilage degeneration were evaluated. RESULTS: Three months after implantation, the implant was completely filled with fibrovascular tissue. After 6 months, the central areas of the implant contained cartilage-like tissue with abundant collagen type II and proteoglycans in their matrix. The foreign-body reaction remained limited to a few giant cells in the implant. The compression modulus of the implant-tissue construct still differed significantly from that of the native meniscus, even at 6 months. Cartilage degeneration was observed both in the meniscectomy group and in the implant group. CONCLUSION: The improved properties of these polymer implants resulted in a faster tissue infiltration and in phenotypical differentiation into tissue resembling that of the native meniscus. However, the material characteristics of the implant need to be improved to prevent degeneration of the articular cartilage. CLINICAL RELEVANCE: The porous polymer implant developed into a polymer-tissue construct that resembled the native meniscus, and with improved gliding characteristics, this prosthesis might be a promising implant for the replacement of the meniscus.     

关节软骨修复细胞基因治疗中软骨细胞支架载体特点

目的 探讨四种不同类型三维支架中培养的关节软骨细胞活性状况及其转基因特性.方法 原代培养兔膝关节软骨细胞,并且以表达绿色荧光蛋白(GFP)和萤火虫荧光素酶(GL3)的腺病毒载体AdGFP和AdGL3进行感染.在I型胶原海绵、纤维蛋白胶、透明质酸和聚乳酸四种类型的支架材料中,以荧光显微镜和荧光素酶分析法检测细胞的活性状况及其转基因特性;以阿辛蓝染色评价软骨基质的产生.结果 表达绿色荧光蛋白的腺病毒(AdGFP)成功感染兔关节软骨细胞,并且持续表达绿色荧光蛋白.被感染的软骨细胞在所有被测试的细胞支架上均能存活,并能表达GFP和荧光素酶报告基因.与其余三种支架比较,聚乳酸支架中转基凶表达率最高(P<0.01).而且聚乳酸培养体系中,4周时可以检测到阿辛蓝染色阳性的基质材料.结论 在关节软骨修复的细胞基因治疗领域,聚乳酸可能是一种合适的支架材料.     

Healing of meniscal tissue by cellular fibrin glue: an in vivo study

Menisci represent fundamental structures for the maintenance of knee homeostasis, playing a key role in knee biomechanics. However, their intrinsic regenerative potential is poor. As a consequence, when a lesion occurs and the meniscus is partially removed by surgery, knee mechanics is subject to dramatic changes. These have been demonstrated to lead often to the development of early osteoarthritis. Therefore, menisci should be repaired whenever possible. In the last decades, tissue engineering approaches have been advocated to improve the reparative processes of joint tissues. In this study, the bonding capacity of an articular chondrocytes-fibrin glue hydrogel was tested as a biologic glue to improve the bonding between two swine meniscal slices in a nude mouse model. The composites were wrapped with acellular fibrin glue and implanted in subcutaneous pouches of nude mice for 4 weeks. Upon retrieval, a firm gross bonding was observed in the experimental samples while none of the control samples, prepared with acellular fibrin glue at the interface, presented any sign of bonding. This was consistent with the histological and scanning electron microscope findings. In particular, a fibrocartilaginous tissue was found at the interface between the meniscal slices, partially penetrating the native meniscus tissue. In order to overcome the lack of regenerative properties of the meniscus, the rationale of using cellular fibrin glue is that fibrin provides immediate stability while carrying cells in the site of lesion. Moreover, fibrin gel is recognized as an optimal scaffold for cell embedding and for promoting fibrocartilaginous differentiation of the cells which synthesize matrix having healing property. These results demonstrated the potential of this model for improving the meniscal bonding. However, further orthotopic studies in a large animal model are needed to evaluate its potential for clinical application.     

Biochemical and biomechanical properties of lesion and adjacent articular cartilage after chondral defect repair in an equine model

BACKGROUND: Chondral defects may lead to degradative changes in the surrounding cartilage, predisposing patients to developing osteoarthritis. PURPOSE: To quantify changes in the biomechanical and biochemical properties of the articular cartilage adjacent to chondral defects after experimental defect repair. STUDY DESIGN: Controlled laboratory study. METHODS: Specimens were harvested from tissue within (lesion), immediately adjacent to, and at a distance from (remote area) a full-thickness cartilage defect 8 months after cartilage repair with genetically modified chondrocytes expressing insulin-like growth factor-I or unmodified, control chondrocytes. Biomechanical properties, including instantaneous Young's and equilibrium aggregate moduli, were determined by confined compression testing. Biochemical properties, such as water and proteoglycan content, were also measured. RESULTS: The instantaneous Young's modulus, equilibrium modulus, and proteoglycan content increased, whereas water content decreased with increasing distance from the repaired lesion. The instantaneous Young's and equilibrium moduli of the adjacent articular cartilage were 80% and 50% that of remote area samples, respectively, whereas water content increased 0.9% and proteoglycan content was decreased by 35%. No significant changes in biomechanical and biochemical properties were found either in the lesion tissue or in adjacent cartilage with genetic modification of the chondrocytes. CONCLUSION: Articular cartilage adjacent to repaired chondral defects showed significant remodeling 8 months after chondral defect repair, regardless of whether genetically modified or unmodified cells were implanted. CLINICAL RELEVANCE: Changes in the biochemical and biomechanical properties of articular cartilage adjacent to repaired chondral defects may represent remodeling as part of an adaptive process or degeneration secondary to an altered distribution of joint forces. Quantification of these changes could provide important parameters for assessing progress after operative chondral defect repair.     

MR imaging of autologous chondrocyte implantation of the knee

Autologous chondrocyte implantation (ACI) is a surgical technique that is increasingly being used in the treatment of full-thickness defects of articular cartilage in the knee. It involves the arthroscopic harvesting and in vitro culture of chondrocytes that are subsequently implanted into a previously identified chondral defect. The aim is to produce a repair tissue that closely resembles hyaline articular cartilage that gradually becomes incorporated, restoring joint congruity. Over the long term, it is hoped that this will prevent the progression of full-thickness articular cartilage defects to osteoarthritis. This article reviews the indications and operative procedure performed in ACI. Magnetic resonance imaging (MRI) sequences that provide optimal visualization of articular cartilage in the post-operative period are discussed. Normal appearances of ACI on MRI are presented along with common complications that are encountered with this technique.     

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．     

组织工程软骨移植物修复兔关节软骨缺损

目的　观察组织工程软骨移植物修复兔关节软骨缺损的效果。　方法　经软骨起源诱导后的兔骨髓间质干细胞(mesenchymalstemcells,MSCs),与牛Ⅰ型胶原及人纤维蛋白相混合制成组织工程软骨移植物。60只5个月龄的日本大耳白兔均分为软骨移植物组、单纯载体对照组和空白对照组,观察各组修复兔股骨髁关节软骨全层缺损的效果。　结果　软骨移植物组12周时已形成正常厚度的软骨层及完整的软骨下骨板,O'drilscoll组织学评分18.22±2.45,Ⅱ型胶原含量97.9%,甲苯胺蓝变色反应表明其与周围正常软骨无明显区别,为透明软骨组织修复。而对照组12周时为纤维软骨修复,后期为纤维组织和板层骨修复。　结论　该组织工程软骨移植物作为软骨移植的替代物是可行的。     

Comparison of fresh osteochondral autografts and allografts: a canine model

BACKGROUND: Osteochondral autografts and allografts have been widely used in the treatment of isolated grade IV articular cartilage lesions of the knee. However, the authors are not aware of any study that has prospectively compared fresh osteochondral autografts to fresh allografts with regard to imaging, biomechanical testing, and histology. HYPOTHESIS: The imaging, biomechanical properties, and histologic appearance of fresh osteochondral autograft and fresh allograft are similar with respect to bony incorporation into host bone, articular cartilage composition, and biomechanical properties. STUDY DESIGN: Controlled laboratory study. METHODS: Eighteen adult dogs underwent bilateral knee osteochondral graft implantation after creation of an Outerbridge grade IV cartilage defect. One knee received an autograft, and the contralateral knee received a fresh allograft. Nine dogs were sacrificed at 3 months, and 9 dogs were sacrificed at 6 months. Graft analysis included gross examination, radiographs, magnetic resonance imaging, biomechanical testing, and histology. RESULTS: Magnetic resonance imaging demonstrated excellent bony incorporation of both autografts and allografts. Biomechanical testing demonstrated no significant difference between autografts versus allografts versus control at 3 or 6 months (P = .36-.91). A post hoc calculation showed 80% power to detect a 30% difference between allograft and control. Histologic examination showed normal cartilage structure for both autografts and allografts. CONCLUSION: Fresh osteochondral autograft and fresh allograft tissues are not statistically different with respect to bony incorporation, articular cartilage composition, or biomechanical properties up to 6 months after implantation. CLINICAL RELEVANCE: The use of fresh allograft tissue to treat osteochondral defects eliminates morbidity associated with harvesting autograft tissue without compromising the results of the surgical procedure.     

软骨细胞在微载体中的培养和快速扩增

目的：探索在短期内获得大量成活率高、分化良好的兔关节软骨细胞的方法,方法：应用胰蛋白酶、胶酶消化的方法从新生新西兰兔关节软骨处分离、培养软骨细胞,并将获得的软骨细胞在旋转生物反应应（RCCS）内应用Cytodex-3微载体进行培养。应用倒置显微镜和扫描电镜对微载体表面的软骨细胞进行动态观察,并对收获的软骨细胞进行Ⅰ、Ⅱ型胶原的细胞免疫化学染色分析。结果：并节软骨细胞可快速贴附于Cytodex-3微载体表面,细胞伸展后生长加速,到培养后期,细胞密度可达最初接种的20倍。在微载体上收获的软骨细胞Ⅰ型胶原的免疫细胞化学染色呈阴性,Ⅱ型胶原染色则呈强阳性。结论：微载体细胞培养技术是一种简便、快速的体外细胞扩增方法,可为构体建组织工程化人工软骨提供大量软骨软件。     

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?     

Contact pressures at grafted cartilage lesions in the knee

The use of tissue-engineered cellular constructs is currently under clinical evaluation for the surgical treatment of articular cartilage lesions in the knee. The primary failure mode in such cartilage repair techniques is related to fixation. In addition, the repair tissue is believed to be very fragile in the post-operative period, and unable to support the intra-articular loads. We have developed a laboratory testing protocol in order to quantify the contact pressure distribution that develops on fibrin glue grafts applied to full-thickness cartilage lesions. The contact pressure distribution has been mapped on the contact surface of specimens subject to compression, in three configurations (intact, defect and grafted), at increasing load levels. All the maps show stress concentrations at the rim of the defect and a more uniform stress distribution around the rim after defect grafting. At a contact load of 180 N, the peak contact pressure measured on cartilage is 2.5 MPa. In presence of the graft, the peak pressures on the cartilage area surrounding the defect are reduced by 16%, on average. In contrast, both the mean contact pressure on the graft and the grafts contact area increase. The graft was found to carry around 80% of the total applied contact load, at all load levels tested. Fibrin glue was chosen as a grafting material in our study because it shows material properties very representative of currently-implanted cellular constructs. Thus, the results of this study have quantified aspects of recipient graft sites that may assist in optimising such grafting procedures from a biomechanical point of view.     

Signalling cascades in mechanotransduction: cell–matrix interactions and mechanical loading

Mechanical loading of articular cartilage stimulates the metabolism of resident chondrocytes and induces the synthesis of molecules to maintain the integrity of the cartilage. Mechanical signals modulate biochemical activity and changes in cell behavior through mechanotransduction. Compression of cartilage results in complex changes within the tissue including matrix and cell deformation, hydrostatic and osmotic pressure, fluid flow, altered matrix water content, ion concentration and fixed charge density. These changes are detected by mechanoreceptors on the cell surface, which include mechanosensitive ion channels and integrins that on activation initiate intracellular signalling cascades leading to tissue remodelling. Excessive mechanical loading also influences chondrocyte metabolism but unlike physiological stimulation leads to a quantitative imbalance between anabolic and catabolic activity resulting in depletion of matrix components. In this article we focus on the role of mechanical signalling in the maintenance of articular cartilage, and discuss how alterations in normal signalling can lead to pathology.     

关节软骨细胞体外培养时生物学性状的变化

<正> 在运动医学领域中,由于运动员的关节软骨伤病发生率远高于普通人,同时又缺乏有效的治疗方法,影响训练和运动成绩的提高,因此对关节软骨伤病的研究一直是重点科研项目之一。对关节软骨进行研究的手段近20年来有了巨大进步,其标志是关节软骨细胞在体外分离培养取得成功。它作为探索软骨细胞性状的先进手段正越来越广泛地被应用到关节软骨损伤和疾患的研究工作中。我所1982年在体外分离培养关节软骨细胞获得成功。近年来,对关节软骨细胞在体外培养过程中所     

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.     

Fresh osteochondral allografting

The treatment of articular cartilage defects in the knee is a difficult challenge. Fresh, small-fragment osteochondralallografting is a technique involving the transplantation of articular (hyaline) cartilage into the defective joint surface. The graft, a composite of living cartilage and a thin layer of underlying subchondral bone, provides a mature matrix with viable chondrocytes along with an osseous component that provides a surface for fixation and integration with the host. Fresh allografting is particularly useful in larger lesions (greater than 2 cms) or when associated osseous defects are present. Clinical experience with fresh osteochondral allografts now extends over 2 decades. Up to 90% of individuals treated for femoral condyle lesions are improved. The allograft tissue appears well tolerated by the host, with documented long-termsurvival of chondrocytes and intact matrix. Successful clinical outcomes have established fresh osteochondrall allografting as an appropriate alternative in the treatment of chondral and osteochondral lesions of the knee.     

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。