Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants.

BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.

HISTORIAL: Suero autólogo acondicionado (ACS) es usado para tartar osteoartritis en caballos, aunque sus efectos no han sido completamente investigados. OBJETIVOS: Investigar los efectos de suero equino y suero acondicionado en condrocitos estimulados con interleukina (IL)-1ß y explantes de cartílago con osteoartritis leve. DISEÑO DEL ESTUDIO: Estudio experimental in vitro. MÉTODOS: El efecto de tres preparaciones séricas diferentes (control no incubado (PS), suero incubado 24 h (PS24h), y suero incubado 24 h en frascos ACS (PCS)) combinados y obtenidos de caballos cojos fueron probados en dos modelos in vitro. Las concentraciones de IL-1ß y de receptor antagonista de IL-1 (IL-1Ra) fueron medidas en todos los sueros. En el modelo 1, los cultivos de pellets de condrocitos fueron estimulados con IL-1ß antes de ser tratados con las preparaciones séricas durante 2 y 48 h. Se realizaron análisis de micromatrices, reacciones de polimerasa en cadena y de matriz de metalopeptidasa-13. En el modelo 2, explantaciones de cartílago proveniente de caballos con osteoartritis estructural fueron tratados con PS o PCS en los días 0, 6 y 12, o dejados sin tartar, y evaluados al día 24 usando la escala de graduación OARSI para evaluación histológica de cartílago articular. RESULTADOS: La concentración de IL-1Ra en PS24h y PCS fue significativamente mayor que en PS. En el modelo 1, los genes relacionados a la inflamación y a la degradación de la matriz cartilaginosa estaban aumentados después de 48 h en todos los grupos tratados en comparación a los controles no tratados. Las moléculas de matriz cartilaginosa, agrecanos y colágenos estaban disminuidos en los pellets PS24h y PCS versus los controles no tratados. Los genes de señales de factores de crecimiento FGF7 estaban aumentados en todos los grupos tratados, BMP2 en PS24h y INHBA in PCS en comparación con los controles no tratados. En el modelo 2, la escala OARSI al día 24 no fue significativamente distinta entre los grupos de tratamientos. LIMITACIONES PRINCIPALES: Los resultados de modelos in vitro no pueden ser directamente aplicados a situaciones in vivo. CONCLUSIONES: El tratamiento in vitro con suero acondicionado no alivió las respuestas inducidas por IL-1ß en pellets de condrocitos o llevo a mejoramiento morfológico en explantes de cartílago con osteoartritis.

Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants.

BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h], and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6, and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS- treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated- compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.

Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink.

Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.

Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.

Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.