The influence of IL-10 and TNFα on chondrogenesis of human mesenchymal stromal cells in three-dimensional cultures.

Chondrogenic differentiated mesenchymal stromal cells (MSCs) are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D) culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL)-10 and Tumor Necrosis Factor (TNF)α on chondrogenesis by MSCs in 3D high-density (H-D) culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA) scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls.

Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model.

Hypothesizing that the implantation of non-articular (heterotopic) chondrocytes might be an alternative approach to support articular cartilage repair, we analyzed joint cartilage defect healing in the rabbit model after implantation of autologous auricle-derived (auricular) chondrocytes. Autologous lapine articular and auricular chondrocytes were cultured for 3 weeks in polyglycolic acid (PGA) scaffolds before being implanted into critical sized osteochondral defects of the rabbit knee femoropatellar groove. Cell-free PGA scaffolds and empty defects served as controls. Construct quality was determined before implantation and defect healing was monitored after 6 and 12 weeks using vitality assays, macroscopical and histological score systems. Neo-cartilage was formed in the PGA constructs seeded with both articular and auricular chondrocytes in vitro and in vivo. At the histological level, cartilage repair was slightly improved when using autologous articular chondrocyte seeded constructs compared to empty defects and was significantly superior compared to defects treated with auricular chondrocytes 6 weeks after implantation. Although only the immunohistological differences were significant, auricular chondrocyte implantation induced an inferior healing response compared with the empty defects. Elastic auricular chondrocytes might maintain some tissue-specific characteristics when implanted into joint cartilage defects which limit its repair capacity.

Hybrid pig versus Gottingen minipig-derived cartilage and chondrocytes show pig line-dependent differences.

Minipigs are widely used as a large animal model for cartilage repair. However, many in vitro studies are based on porcine chondrocytes derived from abundantly available premature hybrid pigs. It remains unclear whether pig line-dependent differences exist which could limit the comparability between in vitro and in vivo results using either hybrid or miniature pig articular chondrocytes. Porcine knee joint femoral cartilage was isolated from 3- to 5-month-old hybrid pigs and Göttingen minipigs. Cartilage from both pig lines was analysed for thickness, zonality, cell content, size and proteoglycan deposition. Cultured articular chondrocytes from both pig lines were investigated for gene and/or protein expression of cartilage-specific proteins such as type II collagen, aggrecan, the chondrogenic transcription factor Sox9, non-specific type I collagen and the cell-matrix receptor ß1-integrin. Cartilage was significantly thinner in the miniature pig compared to the hybrid pig, but the differences between the medial and lateral femur condyles did not reach a significant level. Knee joint cartilage zone formation started only in the minipig, whereas cellularity and cell diameters were comparable in both pig lines. Blood vessels could be detected in the hybrid pig but not the minipig cartilage. Sulphated proteoglycan deposition was more pronounced in cartilage zones II-IV of both pig lines. Minipig chondrocytes expressed type II and I collagen, Sox9 and ß1-integrin at a higher level than hybrid pig chondrocytes. These distinct line-dependent differences should be considered when using hybrid pig-derived chondrocytes for tissue engineering and Göttingen minipigs as a large animal model.

Heterotopic and orthotopic autologous chondrocyte implantation using a minipig chondral defect model.

Implantation of non-articular (heterotopic) chondrocyte-based implants might be an alternative approach to articular cartilage repair. This strategy could be helpful in cases in which there are no or too few articular chondrocytes available. Therefore, this study was undertaken to compare joint cartilage defect healing in the minipig model after implantation of heterotopic auricular and orthotopic articular chondrocytes. Poly-glycolic acid (PGA) associated three-dimensional (3D) constructs were prepared culturing autologous minipig-derived articular and auricular chondrocytes for 7 days in a dynamic culture system. Chondrocyte PGA constructs were implanted into 8mm diameter and ∼1.1mm deep chondral defects within the medial and lateral condyles of the minipig knee joints. Empty defects served as controls for assessment of the intrinsic healing response. Defect healing was monitored 6 months post implantation using a macroscopic and microscopic score system and biomechanical analysis. Neo-cartilage formation could be observed in the PGA constructs seeded with articular and auricular chondrocytes in vivo. The defect healing did not significantly differ at the macroscopic and histological level in response to implantation of either autologous articular or auricular chondrocytes seeded constructs compared with the empty defects. Although the differences were not significant, the auricular chondrocytes-based implants led to a slightly inferior repair quality at the macroscopic level, but a histologically superior healing response when compared with the empty defect group. However, biomechanical analysis revealed a higher stiffness in repair tissues produced by auricular chondrocyte implantation compared with the other groups. Deduced from these results, articular chondrocytes represent the preferable cell source for implantation.

Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix.

Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.

Heterotopic autologous chondrocyte transplantation--a realistic approach to support articular cartilage repair?

Injured articular cartilage is limited in its capacity to heal. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but it requires articular cartilage biopsies for sufficient autologous chondrocyte expansion in vitro. Hence, ACT is restricted by donor-site morbidity and autologous articular chondrocytes availability. The use of nonarticular heterotopic chondrocytes such as auricular, nasoseptal, or costal chondrocytes for ACT might overcome these limitations: heterotopic sources show lesser donor-site morbidity and a comparable extracellular cartilage matrix synthesis profile to articular cartilage. However, heterotopic (h)ACT poses a challenge. Particular tissue characteristics of heterotopic cartilage, divergent culturing peculiarities of heterotopic chondrocytes, and the advantages and drawbacks related to these diverse cartilage sources were critically discussed. Finally, available in vitro and in vivo experimental (h)ACT approaches were summarized. The quality of the cartilage engineered using heterotopic chondrocytes remains partly controversy due to the divergent methodologies and culture conditions used. While some encouraging in vivo results using (h)ACT have been demonstrated, standardized culturing protocols are strongly required. However, whether heterotopic chondrocytes implanted into joint cartilage defects maintain their particular tissue properties or can be adapted via tissue engineering strategies to fulfill regular articular cartilage functions requires further studies.

Characterization of auricular chondrocytes and auricular/articular chondrocyte co-cultures in terms of an application in articular cartilage repair.

Cartilage injury remains a challenge in orthopedic surgery as articular cartilage only has a limited capacity for intrinsic healing. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but requires articular cartilage biopsies for autologous chondrocyte expansion. The use of heterotopic chondrocytes derived from non-articular cartilage sources such as auricular chondrocytes may be a novel approach for ACT. The aim of the study is to evaluate whether co-cultured articular/auricular chondrocytes exhibit characteristics comparable to articular chondrocytes. Analysis of the proliferation rate, extracellular cartilage matrix (ECM) gene and protein expression (type II and I collagen, elastin, lubricin), beta1-integrins and the chondrogenic transcription factor sox9 in articular/auricular chondrocytes was performed using RTD-PCR, flow cytometry, immunofluorescence microscopy and Western blot analysis. Additionally, three-dimensional (3D) chondrocyte mono- and co-cultures were established. The proliferative activity and elastin gene expression were lower and that of type II collagen and lubricin was higher in articular compared with auricular chondrocytes. The species generally did not influence the chondrocyte characteristics, with the exception of type I collagen and sox9 expression, which was higher in porcine but not in human articular chondrocytes compared with both types of auricular chondrocytes. beta1-integrin gene expression did not differ significantly between the chondrocyte types. The type II collagen gene and protein expression was higher in articular chondrocyte monocultures and was slightly higher in co-cultures compared with monocultured auricular chondrocytes. Both chondrocyte types survived in co-culture. Despite their differing expression profiles, co-cultures revealed some adjustment in the ECM expression of both chondrocyte types.

Laser-assisted cholesteatoma surgery: technical aspects, in vitro implementation and challenge of selective cell destruction.

Cholesteatoma is a destructive ear condition requiring complete surgical removal. One major problem lies in the frequent occurrence of residual cholesteatoma caused by squamous epithelium remaining in the middle ear. Our aim is to develop a laser treatment that is selectively directed against residual cholesteatoma cells and can be performed after cholesteatoma surgery in the same session. In a first trial, we studied the photodynamic effect of argon (AL) and diode lasers (DL) on cholesteatoma tissue. Intraoperatively harvested monolayer-cultured cholesteatoma cells were stained in vivo with different absorption enhancers: neutral red (NR), fluorescein diacetate (FDA), and indocyanine green (ICG). In vitro, staining tests on enhanced cellular dye absorption and laser tests were followed by cytotoxicity measurements to determine the respective amount of damage. To achieve selective cell destruction, antibody-mediated staining of cholesteatoma and middle ear mucosa cells was examined in a second trial. Cell cultures (cytospin and coverglass growing) and paraffin-embedded cholesteatoma tissue sections were studied immunohistochemically to determine the binding of monoclonal mouse antibodies against human cytokeratins CK5, CK10, CK14 and the epidermal growth factor receptor EGFR. Intracellular staining with absorption enhancers increased the optical density at the wavelength corresponding to the dye. Staining and subsequent laser irradiation destroyed up to 92% of cultured cholesteatoma cells. Unstained irradiated tissue was not affected. In cytospins, the antibody against CK5/6 showed strong staining of cholesteatoma and weak staining of mucosa cells. Reactivity for CK14 and EGFR was positive in both tissues. In coverglass cultures, staining of cholesteatoma cells was positive for CK5/6, CK14 and EGFR. Mucosa cells were positive for EGFR but negative for cytokeratins. Both cell types were negative for CK10. In embedded cholesteatoma tissue, CK5/6 and CK14 were localized in the basal layers of the matrix, while CK10 was situated in the suprabasal layers, and EGFR was present in all layers of the matrix and perimatrix. As for the technical aspects of laser-assisted cholesteatoma surgery, AL and DL have proved to be suitable devices; ICG and FDA are effective nontoxic absorption enhancers. The investigated antibodies against cytokeratins and EGFR show nonselective staining and thus appear to be inappropriate for avoiding unwanted cell damage. For safe and specific intraoperative application to intact tissue, the chromophore should be coupled to a particular antibody that binds solely to an easily accessible specific antigen at the surface of cholesteatoma cells.