A novel DNA profiling application for the monitoring of cross-contamination in autologous chondrocyte implantation.

BACKGROUND: Autologous chondrocyte implantation (ACI) is a cell-based treatment that can be used to regenerate chondral defects. European legislation specifically classifies such produced chondrocytes as "medicinal for advanced cell therapy" that have to be manufactured in pharmaceutical factories according to specific rules, named Good Manufacturing Practices (GMPs). One main requirement of cell manipulation in advanced therapy is to prevent the risk of any contamination. AIM: The aim of this study was to verify if chondrocyte cultures suitable for ACI were free of cross-contamination by means of DNA profiling techniques. MATERIALS AND METHODS: Cell cultures were carried on in a Hospital Cell Factory in compliance with European current Good Manufacturing Practices. DNA profiling, by means of Short Tandem Repeats and miniShort Tandem Repeats analyses, was performed on expanded chondrocytes and their related control blood samples. Mitochondrial DNA was analysed to further confirm the results and to evaluate possible mutations occurred in the samples. RESULTS: Our findings demonstrated the absence of cross-contamination between chondrocyte cultures and, thus, their identity maintenance until the end of the manipulation. CONCLUSIONS: DNA profiling technique can be a suitable test for quality control not only for chondrocyte manipulation, but for cell therapy in general.

Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold.

OBJECTIVES: Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. METHODS: A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. RESULTS: This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. CONCLUSION: This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci.Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101-106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.

Ligament repair: a molecular and immunohistological characterization.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.

Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits.

Different methods have been used to improve chondrocyte transplantation for the repair of articular cartilage defects. Several groups of biomaterials have been proposed as support for in vitro cell growth and for in vivo implantation. Here. we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a hyaluronan derivative referred to as Hyaff-11. Full thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histologic samples from in and around the defect sites were examined 1, 3 and 6 months after surgery and were scored from 0 to 16. Statistically significant differences in the quality of the regenerated tissue were found between the grafts carried out with biomaterial carrying chondrocyte cells compared to the biomaterial alone or controls. This study demonstrates the efficacy of this hyaluronan-based scaffold for autologous chondrocytes transplantation.

Expression of different chemokines by human osteosarcoma cells in response to tumor necrosis factor-alpha.

We investigated the expression of different chemokines in the surnatants and inside the cells of four human osteosarcoma cell lines. HOS, U-2 OS, MG63 and Saos-2 cells were cultured for 24, 48, 72 hours both in basal conditions and after stimulus with TNF alpha. Human stromal cells were used as control. IL-8 and MCP-1 are present in higher concentration in the surnatants in contrast to RANTES which is present primarily inside the cells. IL-8 and MCP-1 are not totally expressed by all the human osteosarcoma cell lines in unstimulated conditions, but became detectable after TNF alpha treatment. In general, this cytokine stimulated the production and release of the three chemokines.

[Tissue engineering applications: cartilage lesions repair by the use of autologous chondrocytes]. / Applicazioni dell'ingegneria tissutale: riparazione di lesioni cartilaginee con condrociti autologhi.

Promising new therapies based on tissue engineering have been recently developed for cartilage repair. The association of biomaterials with autologous chondrocytes expanded in vitro can represent a useful tool to regenerate this tissue. The scaffolds utilised in such therapeutical applications should provide a pre-formed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells and stimulate the phenotype of transplanted cells. Hyaff-11 is a hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell carrier for tissue-engineered repair. From our findings we can state that human chondrocytes seeded on Hyaff-11 are able to maintain in vitro the characteristic of differentiated cells, expressing and producing collagen type II and aggrecan which are the main markers of cartilage phenotype, down-regulating collagen type I. Moreover, it seems to be a useful scaffold for cartilage repair both in animal models and clinical trials in humans, favouring the formation of a hyaline-like tissue. In the light of these data, we can hypothesise, for the future, the use of autologous chondrocyte transplantation together with gene therapy as a treatment for rheumatic diseases such as osteoarthritis.

Establishment of a new human immortalized chondrocyte cell line.

Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorescence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium and seeding on a hyaluronan-based three-dimensional biomaterial. Immortalized cells were able to re-express the main markers of chondrocyte phenotype, both at mRNA and protein levels, under the two defined cultured conditions used. The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation, and of the etiopathogenesis of many rheumatic diseases.

Tissue engineering for cartilage repair: in vitro properties of a hyaluronan-derivative.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a performed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaff-11 is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaff-11 to support the growth of human chondrocytes and to maintain their original phenotype. Our data indicate that human chondrocytes seeded on Hyaff-11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration of therapeutic potential of Hyaff-11 as a delivery vehicle in tissue-engineered repair of articular cartilage defects.

In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications.

OBJECTIVE: A number of studies have shown the role of expanded Bone Marrow-derived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

A molecular and histological characterization of cartilage from patients with Morquio syndrome.

OBJECTIVE: To investigate the gene expression profile and the histological aspects of articular cartilage of patients affected by Morquio syndrome, a lysosomal storage disease characterized by the accumulation of glycosaminoglycans within the cells which result in abnormal formation and growth of the skeletal system. METHOD: Articular cartilage samples were obtained from the femoral condyle of two siblings with Morquio syndrome during surgery performed to treat valgus knee. As controls, four biopsy samples of healthy cartilage were obtained from four different male multiorgan donors. A Real-Time Polymerase Chain reaction (RT-PCR) analysis was performed to evaluate the expression of type I and II collagens and aggrecan mRNAs. Histological and immunohistochemical analyses for some matrix proteins were carried out on paraffin embedded sections. RESULTS: Type I collagen mRNA mean level was higher in the samples of patients with Morquio syndrome compared to controls. Type II collagen and aggrecan mRNAs' mean expression was instead lower. The morphological appearance of the cartilage showed a poorly organized tissue structure with not homogeneously distributed cells that were larger compared to normal chondrocytes due to the presence inside the vacuoles of proteoglycans which were not metabolized. Chondrocytes were negative for collagen II immunostaining while the extracellular matrix was weakly positive. Collagen type I immunostaining was positive at cellular level. Keratan sulfate showed diffuse positivity and chondroitin-6-sulfate was present throughout the cartilaginous thickness. CONCLUSION: In cartilage of patients with Morquio syndrome, a low expression of collagen type II and a high expression of collagen type I both at protein and molecular levels are evidentiated. This finding could give evidence of the reduction in ankle and knee joint movement observable in these patients.

Differential roles of nitric oxide and oxygen radicals in chondrocytes affected by osteoarthritis and rheumatoid arthritis.

Osteoarthritis and rheumatoid arthritis are characterized by focal loss of cartilage due to an up-regulation of catabolic pathways, induced mainly by pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor alpha (TNFalpha). Since reactive oxygen species are also involved in this extracellular-matrix-degrading activity, we aimed to compare the chondrocyte oxidative status responsible for cartilage damage occurring in primarily degenerative (osteoarthritis) and inflammatory (rheumatoid arthritis) joint diseases. Human articular chondrocytes were isolated from patients with osteoarthritis or rheumatoid arthritis, or from multi-organ donors, and stimulated with IL-1beta and/or TNFalpha. We evaluated the oxidative stress related to reactive nitrogen and oxygen intermediates, measuring NO(-)(2) as a stable end-product of nitric oxide generation and superoxide dismutase as an antioxidant enzyme induced by radical oxygen species. We found that cells from patients with osteoarthritis produced higher levels of NO(-)(2) than those from patients with rheumatoid arthritis. In addition, IL-1beta was more potent than TNFalpha in inducing nitric oxide in both arthritides, and TNFalpha alone was almost ineffective in cells from rheumatoid arthritis patients. We also observed that the intracellular content of copper/zinc superoxide dismutase (Cu/ZnSOD) was always lower in rheumatoid arthritis chondrocytes than in those from multi-organ donors, whereas no differences were found in intracellular manganese SOD (MnSOD) or in supernatant Cu/ZnSOD and MnSOD levels. Moreover, intracellular MnSOD was up-regulated by cytokines in osteoarthritis chondrocytes. In conclusion, our results suggest that nitric oxide may play a major role in altering chondrocyte functions in osteoarthritis, whereas the harmful effects of radical oxygen species are more evident in chondrocytes from patients with rheumatoid arthritis, due to an oxidant/antioxidant imbalance.

Human articular chondrocytes immortalized by HPV-16 E6 and E7 genes: Maintenance of differentiated phenotype under defined culture conditions.

OBJECTIVE: To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases. DESIGN: Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification. RESULTS: Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes. CONCLUSIONS: The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans.

White cell apoptosis in platelet concentrates.

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, and tumor necrosis factor alpha). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BC-PCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.