Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells

Focal articular cartilage damage can eventually lead to the onset of osteoarthritis with degradation around healthy articular cartilage. Currently, there are no drugs available that effectively repair articular cartilage damage. Several surgical techniques exist and are expected to prevent progression to osteoarthritis, but they do not offer a long‐term clinical solution. Recently, regenerative medicine approaches using human pluripotent stem cells (PSCs) have gained attention as new cell sources for therapeutic products. To translate PSCs to clinical application, appropriate cultures that produce large amounts of chondrocytes and hyaline cartilage are needed. So too are assays for the safety and efficacy of the cellular materials in preclinical studies including animal transplantation models. To confirm safety and efficacy, transplantation into the subcutaneous space and articular cartilage defects have been performed in animal models. All but one study we reviewed that transplanted PSC‐derived cellular products into articular cartilage defects found safe and effective recovery. However, for most of those studies, the quality of the PSCs was not verified, and the evaluations were done with small animals over short observation periods. Large animals and longer observation times are preferred. We will discuss the recent progress and future direction of the animal transplantation studies for the treatment of focal articular cartilage damages using PSCs.     

Toward regeneration of articular cartilage

Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra‐articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. Birth Defects Research (Part C) 99:192–202, 2013 . © 2013 Wiley Periodicals, Inc .     

深低温冻存组织工程化软骨在关节软骨缺损修复的应用

目的：探讨经深低温冻存组织工程化软骨修复关节软骨缺损的可行性。方法：分离收集3周龄新西兰大白兔关节软骨细胞进行体外培养,接种于PGA三维支架材料上,复合物体外培养1周后冻存,冻存1个月后解冻、复苏及体外培养,1周后接种于已建立的双侧兔膝关节软骨缺损模型的膝关节软骨缺损处,并设对照组。分别于手术后4周、8周、12周行大体标本及组织观察。结果：大体观察结果表明,实验组与对照组缺损处均由软骨组织修复;组织学观察可以见到实验组和对照组关节软骨缺损处有密集的软骨细胞,均有软骨生成及基质分泌,两组差异无统计学意义。结论：应用深低温冻存组织工程化软骨修复关节软骨缺损的方法是有效可行的,为其进一步临床应用提供了实验依据。     

Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.Key words: in vitro cartilage, 3-D cell culture, fusion culture technique, tissue engineering, cell differentiation, extracellular matrix, immunohistochemistry     

Chondrocyte suspension in fibrin glue

Autologous chondrocyte implantation has been shown to be a promising method for treatment of deep articular cartilage defects. The hyaline cartilage formed by implanted autologous chondrocytes has biomechanical properties similar to those of natural articular cartilage. Between June 2006 and September 2008 we performed Autologous chondrocyte implantation (ACI) in 50 patients and the chondrocytes were supported in fibrin glue. The cartilage biopsy samples were taken from the non-weight bearing area of the patient’s femoral condyle and the samples were transferred to the cell culture laboratory. Chondrocyte were kept in culture about 20 days. Fibrin glue was used as a three dimensional carrier for chondrocyte implantation. A 450 ml of patient’s own blood was collected prior to transplantation to produce autologous fibrinogen. Alternatively the allogenic fibrinogen was prepared from Regional Blood Center voluntary donors. Before surgery the chondrocyte suspension was mixed with fibrin glue and gel—like fibrograft was prepared. The total number of cells and the size of fibrograft depended on the defect size in the knee. Our results suggest that ACI technique with fibrin glue is a promising method for treatment of cartilage defect.     

Growth factors and chondrogenic differentiation of mesenchymal stem cells

The main purpose of the article is to review recent knowledge about growth factors and their effect on the chondrogenic differentiation of mesenchymal stem cells under in vitro conditions. Damaged or lost articular cartilage leads to progressive debilitation, which have major impact on the life quality of the affected individuals of both sexes in all age groups. Mature hyaline cartilage has a very low self-repair potential due to intrinsic properties - lack of innervation and vascular supply. Another limiting factor is low mitotic potential of chondrocytes. Small defects are healed by migration of chondrocytes, while large ones are healed by formation of inferior fibrocartilage. However, in many cases osteoarthritis develops. Recently, cellular therapy combining mesenchymal stem cells and proper differentiation factors seems to be promising tool for hyaline cartilage defects healing.     

Requirement of fibroblast growth factor signaling for regeneration of epiphyseal morphology in rabbit full-thickness defects of articular cartilage

The involvement of fibroblast growth factor-2 (FGF-2) during the repair process in rabbit full-thickness defects of articular cartilage was studied. Fibroblast growth factor-2 (50 pg/h) was administered for 2 weeks in a 5mm defect of articular cartilage, which is large enough not to repair spontaneously. The administration of FGF-2 resulted in the regeneration of the articular cartilage and the subchondral bone within 8 weeks. In these defects, undifferentiated mesenchymal cells initiated chondrogenic differentiation coupled with replacement by subchondral bone, resulting in the resurfacing of the defects with hyaline cartilage and the recovery of subchondral bone up to the original bone–articular cartilage junction. In rabbits, full-thickness defects are capable of regenerating articular cartilage as long as the defect size is limited to ≤3 mm in diameter. In the defects, strong immunoreactivity for FGF-2 was observed in the granulation tissue filling the defects in the early stage of repair, in association with the expression of FGF-2 mRNA shown by in situ hybridization. Once the undifferentiated mesenchymal cells had differentiated into chondrocytes, both the immunoreactivity and the in situ hybridization signal declined significantly. Upon the local administration of a monoclonal antibody against FGF-2 (bFM-1, 50ng/h), the defects were filled with fibrous tissue and no resurfacing hyaline cartilage was formed. Compared to the non-treated defects, there were marked increases in FGF-2 immunoreactivity and the overexpression of FGF-2 mRNA in the reparative tissue in the bFM-1 -treated defects. This rebound phenomenon indicates that the autocrine FGF-2 signaling is critically important for the regeneration of articular cartilage.     

Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF-2) in vivo

BACKGROUND: Defects of articular cartilage are an unsolved problem in orthopaedics. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. METHODS: Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cartilage defects in the knee joints of rabbits. In vivo, cartilage repair was assessed qualitatively and quantitatively at 3 and 14 weeks after implantation. RESULTS: In vitro, bioactive FGF-2 was secreted, leading to a significant increase in the cell numbers in FGF-2 spheres. In vivo, FGF-2 continued to be expressed for at least 3 weeks without leading to differences in FGF-2 concentrations in the synovial fluid between treatment groups. Histological analysis revealed no adverse pathologic effects on the synovial membrane at any time point. FGF-2 gene transfer enhanced type II collagen expression and individual parameters of chondrogenesis, such as the cell morphology and architecture of the new tissue. Overall articular cartilage repair was significantly improved at both time points in vivo. CONCLUSIONS: The data suggest that localized overexpression of FGF-2 enhances the repair of cartilage defects via stimulation of chondrogenesis, without adverse effects on the synovial membrane. These results may lead to the development of safe gene-based therapies for human articular cartilage defects.     

Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results

Autologous chondrocyte implantation (ACI) is an effective clinical procedure for the regeneration of articular cartilage defects. BioSeed-C is a second-generation ACI tissue engineering cartilage graft that is based on autologous chondrocytes embedded in a three-dimensional bioresorbable two-component gel-polymer scaffold. In the present prospective study, we evaluated the short-term to mid-term efficacy of BioSeed-C for the arthrotomic and arthroscopic treatment of posttraumatic and degenerative cartilage defects in a group of patients suffering from chronic posttraumatic and/or degenerative cartilage lesions of the knee. Clinical outcome was assessed in 40 patients with a 2-year clinical follow-up before implantation and at 3, 6, 12, and 24 months after implantation by using the modified Cincinnati Knee Rating System, the Lysholm score, the Knee injury and Osteoarthritis Outcome Score, and the current health assessment form (SF-36) of the International Knee Documentation Committee, as well as histological analysis of second-look biopsies. Significant improvement (p < 0.05) in the evaluated scores was observed at 1 and/or 2 years after implantation of BioSeed-C, and histological staining of the biopsies showed good integration of the graft and formation of a cartilaginous repair tissue. The Knee injury and Osteoarthritis Outcome Score showed significant improvement in the subclasses pain, other symptoms, and knee-related quality of life 2 years after implantation of BioSeed-C in focal osteoarthritic defects. The results suggest that implanting BioSeed-C is an effective treatment option for the regeneration of posttraumatic and/or osteoarthritic defects of the knee.     

Quantitative ultrasound can assess the regeneration process of tissue-engineered cartilage using a complex between adherent bone marrow cells and a three-dimensional scaffold

Articular cartilage (hyaline cartilage) defects resulting from traumatic injury or degenerative joint disease do not repair themselves spontaneously. Therefore, such defects may require novel regenerative strategies to restore biologically and biomechanically functional tissue. Recently, tissue engineering using a complex of cells and scaffold has emerged as a new approach for repairing cartilage defects and restoring cartilage function. With the advent of this new technology, accurate methods for evaluating articular cartilage have become important. In particular, in vivo evaluation is essential for determining the best treatment. However, without a biopsy, which causes damage, articular cartilage cannot be accurately evaluated in a clinical context. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the cartilage are measured by introducing an ultrasonic probe during arthroscopy of the knee joint. The purpose of the current study was to determine the efficacy of this ultrasound system for evaluating tissue-engineered cartilage in an experimental model involving implantation of a cell/scaffold complex into rabbit knee joint defects. Ultrasonic echoes from the articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the percentage maximum magnitude (the maximum magnitude of the measurement area of the operated knee divided by that of the intact cartilage of the opposite, nonoperated knee; %MM) was used as a quantitative index of cartilage regeneration. Using this index, the tissue-engineered cartilage was examined to elucidate the relations between ultrasonic analysis and biochemical and histological analyses. The %MM increased over the time course of the implant and all the hyaline-like cartilage samples from the histological findings had a high %MM. Correlations were observed between the %MM and the semiquantitative histologic grading scale scores from the histological findings. In the biochemical findings, the chondroitin sulfate content increased over the time course of the implant, whereas the hydroxyproline content remained constant. The chondroitin sulfate content showed a similarity to the results of the %MM values. Ultrasonic measurements were found to predict the regeneration process of the tissue-engineered cartilage as a minimally invasive method. Therefore, ultrasonic evaluation using a wavelet map can support the evaluation of tissue-engineered cartilage using cell/scaffold complexes.     

Articular Cartilage Chondrocytes are more Advantageous for Generating Hyaline-like Cartilage than Mesenchymal Cells Isolated from Microfracture Repairs

Articular cartilage lacks self-repair capacity. Currently, two methods employing autologous cells are used to stimulate repair of articular cartilage. Micro-fracture induced repair induces autologous mesenchymal cell migration from bone marrow. Autologous chondrocytes' transplantation involves in vitro expansion of chondrocytes, and later implantation. In 15 patients de-differentiated chondrocytes obtained by cartilage biopsy were compared to cells derived from repair tissue induced by micro-fracture. These patients all underwent micro-fracture during the cartilage biopsy procedure. Autologous chondrocytes' transplantation was performed at least two months later then the biopsy. Tissue bits from articular cartilage and micro-fracture repair tissue were incubated in-vitro and explant cell cultures established. The cell cultures were assessed by immunohistochemistry and induced to differentiate. Differentiation into bone tissue was stimulated by addition of basic fibroblast growth factor, ascorbate and dexamethasone. High density (micro-mass) culture was used to stimulate chondrogenesis. Both cell cultures consist of mesenchymal progenitors as indicated by fibroblast growth factor receptor 3 expression and anti-CD-34+ antibodies. However, the micro-fracture generated repair tissue consists of osteocalcin-expressing cells destined to become bone. Collagen type II expression does not occur in these cells compared to autologous chondrocytes. Inducible nitric oxide synthase expression by microfracture cells is likely to damage surrounding articular cartilage in vivo. In conclusion, cells recruited by micro-fracture are inferior for cartilage regeneration purposes to those from cartilage biopsies.     

In vitro effect of a synthesized sulfonamido-based gallate on articular chondrocyte metabolism

Autologous chondrocyte implantation (ACI) is a promising strategy for cartilage repair and reconstitution. However, limited cell numbers and the dedifferentiation of chondrocytes present major difficulties to the success of ACI therapy. Therefore, it is important to find effective pro-chondrogenic agents that restore these defects to ensure a successful therapy. In this study, we synthesized a sulfonamido-based gallate, namely N-[4-(4,6-dimethyl-pyrimidin-2-ylsulfamoyl)-phenyl]-3,4,5-trihydroxy-benzamide (EJTC), and investigated its effects on rabbit articular chondrocytes through an examination of its specific effects on cell proliferation, morphology, viability, GAG synthesis, and cartilage-specific gene expression. The results show that EJTC can effectively promote chondrocyte growth and enhance the secretion and synthesis of cartilage ECM by upregulating the expression levels of the aggrecan, collagen II, and Sox9 genes. The expression of the collagen I gene was effectively downregulated, which indicates that EJTC inhibits chondrocytes dedifferentiation. Chondrocyte hypertrophy, which may lead to chondrocyte ossification, was also undetectable in the EJTC-treated groups. The recommended dose of EJTC ranges from 3.125 μg/mL to 7.8125 μg/mL, and the most profound response was observed with 7.8125 μg/mL. This study may provide a basis for the development of a novel agent for the treatment of articular cartilage defects.     

Mesenchymal stem cells as a potent cell source for articular cartilage regeneration

Since articular cartilage possesses only a weak capac-ity for repair, its regeneration potential is considered one of the most important challenges for orthopedic surgeons. The treatment options, such as marrow stimulation techniques, fail to induce a repair tissue with the same functional and mechanical properties of native hyaline cartilage. Osteochondral transplantation is considered an effective treatment option but is as-sociated with some disadvantages, including donor-site morbidity, tissue supply limitation, unsuitable mechani-cal properties and thickness of the obtained tissue. Although autologous chondrocyte implantation results in reasonable repair, it requires a two-step surgical pro-cedure. Moreover, chondrocytes expanded in culture gradually undergo dedifferentiation, so lose morpho-logical features and specialized functions. In the search for alternative cells, scientists have found mesenchymal stem cells(MSCs) to be an appropriate cellular mate-rial for articular cartilage repair. These cells were origi-nally isolated from bone marrow samples and further investigations have revealed the presence of the cells in many other tissues. Furthermore, chondrogenic dif-ferentiation is an inherent property of MSCs noticedat the time of the cell discovery. MSCs are known to exhibit homing potential to the damaged site at which they differentiate into the tissue cells or secrete a wide spectrum of bioactive factors with regenerative proper-ties. Moreover, these cells possess a considerable im-munomodulatory potential that make them the general donor for therapeutic applications. All of these topics will be discussed in this review.     

Involvement of ATP,increase of intracellular calcium and the early expression of <Emphasis Type="Italic">c-fos</Emphasis> in the repair of rat fetal articular cartilage

To compare the potential of adult and fetal animals to repair articular cartilage, we investigated the early process after creating superficial defects in the femoral knee cartilage in rat models. In fetuses at 19 days of gestation, both chondrocytes and the extracellular matrix responded notably by 48 h after artificial injury. Staining patterns with safranin O revealed that, by 1 h after injury, some components of the extracellular matrix around the wound were modified, and the change spread from the limited region to the entire knee cartilage within 24 h. The chondrocytes in the area surrounding the wound transiently expressed increased level of c-fos from 1 h to 6 h. The wound remained 1 day after birth, i.e., 72 h after injury, but was completely repaired 10 days after birth. In contrast, neither visible responses nor transient c-fos expression was observed in 12-week-old adult articular cartilage 48 h after injury. We also examined the relationships between the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the induction of c-fos expression in the cartilage. Applications of ATP or Ca²⁺ ionophore A23187, both of which increase [Ca²⁺]_i, induced immediate expression of c-fos in primary cultured chondrocytes: 1 M ATP elicited an increase of [Ca²⁺]_i in chondrocytes in fetal cartilage slices, but 1 mM was required in adult cartilage slices. Our findings show the presence of a signaling pathway that is apparently active in the repair of fetal but not adult articular cartilage and that involves the intercellular transfer of ATP, increase of [Ca²⁺]_i, and expression of c-fos in cartilage.This study was supported in part by Health Sciences Research Grants for Research on Human Genome, Tissue Engineering and Food Biotechnology to M.O. from the Japanese Ministry of Health, Labor and Welfare     

Treatment of focal degenerative cartilage defects with polymer-based autologous chondrocyte grafts: four-year clinical results

Introduction

Second-generation autologous chondrocyte implantation with scaffolds stabilizing the grafts is a clinically effective procedure for cartilage repair. In this ongoing prospective observational case report study, we evaluated the effectiveness of BioSeed^®-C, a cell-based cartilage graft based on autologous chondrocytes embedded in fibrin and a stable resorbable polymer scaffold, for the treatment of clinical symptomatic focal degenerative defects of the knee.

Methods

Clinical outcome after 4-year clinical follow-up was assessed in 19 patients with preoperatively radiologically confirmed osteoarthritis and a Kellgren-Lawrence score of 2 or more. Clinical scoring was performed before implantation of the graft and 6, 12, and 48 months after implantation using the Lysholm score, the Knee injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee (IKDC) score, and the International Cartilage Repair Society (ICRS) score. Cartilage regeneration and articular resurfacing were assessed by magnetic resonance imaging (MRI) 4 years after implantation of the autologous cartilage graft.

Results

Significant improvement (P < 0.05) of the Lysholm and ICRS scores was observed as early as 6 months after implantation of BioSeed^®-C and remained stable during follow-up. The IKDC score showed significant improvement compared with the preoperative situation at 12 and 48 months (P < 0.05). The KOOS showed significant improvement in the subclasses pain, activities of daily living, and knee-related quality of life 6 months as well as 1 and 4 years after implantation of BioSeed^®-C in osteoarthritic defects (P < 0.05). MRI analysis showed moderate to complete defect filling with a normal to incidentally hyperintense signal in 16 out of 19 patients treated with BioSeed^®-C. Two patients without improvement in the clinical and MRI scores received a total knee endoprosthesis after 4 years.

Conclusions

The results show that the good clinical outcome achieved 1 year after implantation of BioSeed^®-C remains stable over the course of a period of 4 years and suggest that implanting BioSeed^®-C is a promising treatment option for the repair of focal degenerative defects of the knee.     

Mesenchyme-specific Knockout of ESET Histone Methyltransferase Causes Ectopic Hypertrophy and Terminal Differentiation of Articular Chondrocytes

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

A second-generation autologous chondrocyte implantation approach to the treatment of focal articular cartilage defects

Autologous chondrocyte implantation (ACI) is the most widely used cell-based surgical procedure for the repair of articular cartilage defects. Challenges to successful ACI outcomes include limitation in defect size and geometry as well as inefficient cell retention. Second-generation ACI procedures have thus focused on developing three-dimensional constructs using native and synthetic biomaterials. Clinically significant and satisfactory results from applying autologous chondrocytes seeded in fibrin within a biodegradable polymeric material were recently reported. In the future, third-generation cell-based articular cartilage repair should focus on the use of chondroprogenitor cells and biofunctionalized biomaterials for more extensive and permanent repair.