Notochordal cell-secreted factors stimulate matrix production by canine nucleus pulposus cells and bone marrow derived stromal cells

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Принцип коеволюції в контексті ноосферної концепції В.Вернадського

Досліджується принцип коеволюції людини та природи в контексті ноосферного вчення В.Вернадського. Аналізуються особливості світогляду суспільства ноосферної цивілізації.Исследуется принцип коэволюции человека и природы в контексте ноосферного учения В.Вернадского. Анализуются особенности мировоззрения общества ноосферной цивилизации.The principle of coevolution of person and nature is investigated in the context of noospherian doctrine of V.Vernadsky. The particularities of world view оf society of noospherian civilization are analyzed

The lower in vitro chondrogenic potential of canine adipose tissue-derived mesenchymal stromal cells (MSC) compared to bone marrow-derived MSC is not improved by BMP-2 or BMP-6

Mesenchymal stromal cells (MSC) are used for cell-based treatment for canine osteoarthritis (OA). Compared with human MSCs, detailed information on the functional characterisation of canine MSCs is limited. In particular, the chondrogenic differentiation of canine adipose tissue-derived MSCs (cAT-MSCs) is challenging. In this study, we aimed to compare cAT-MSCs with bone marrow-derived MSCs (cBM-MSCs), focusing specifically on their in vitro chondrogenic potential, with or without bone morphogenetic proteins (BMP). cBM-MSCs and cAT-MSCs were characterised using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chondrogenic differentiation potential of all cMSC preparations in the presence of TGF-β1 alone or when supplemented with 10, 100, or 250 ng/mL BMP-2 or BMP-6 was investigated using RT-qPCR, and biochemical, histochemical and immunohistological analyses. Both cBM-MSCs and cAT-MSCs expressed the surface markers CD90, CD73, and CD29, and were negative for CD45 and CD34, although the expression of CD73 and CD271 varied with donor and tissue origin. Interestingly, expression of ACAN and SOX9 was higher in cBM-MSCs than cAT-MSCs. In contrast with cBM-MSCs, cAT-MSCs could not differentiate toward the chondrogenic lineage without BMP-2/-6, and their in vitro chondrogenesis was inferior to cBM-MSCs with BMP-2/-6. Thus, cAT-MSCs have lower in vitro chondrogenic capacity than cBM-MSC under the studied culture conditions with 10, 100, or 250 ng/mL BMP-2 or BMP-6. Therefore, further characterisation is necessary to explore the potential of cAT-MSCs for cell-based OA treatments

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies

The catabolic-to-anabolic shift seen in the canine osteoarthritic cartilage treated with knee joint distraction occurs after the distraction period

Background Cartilage regenerative mechanisms initiated by knee joint distraction (KJD) remain elusive. Animal experiments that are representative for the human osteoarthritic situation and investigate the effects of KJD at consecutive time points could be helpful in this respect but are lacking. This study investigated the effects of KJD on the osteoarthritic joint of dogs on two consecutive timepoints. Methods Osteoarthritis was bilaterally induced for 10 weeks in 12 dogs using the groove model. Subsequently, KJD was applied to the right hindlimb for 8 weeks. The cartilage, subchondral bone and synovial membrane were investigated directly after KJD treatment, and after 10 weeks of follow-up after KJD treatment. Macroscopic and microscopic joint tissue alterations were investigated using the OARSI grading system. Additionally, proteoglycan content and synthesis of the cartilage were assessed biochemically. RT-qPCR analysis was used to explore involved signaling pathways. Results Directly after KJD proteoglycan and collagen type II content were reduced accompanied by decreased proteoglycan synthesis. After 10 weeks of follow-up, proteoglycan and collagen type II content were partly restored and proteoglycan synthesis increased. RT-qPCR analysis of the cartilage suggests involvement of the TGF-β and Notch signalling pathways. Additionally, increased subchondral bone remodelling was found at 10 weeks of follow-up. Conclusion While the catabolic environment in the cartilage is still present directly after KJD, at 10 weeks of follow-up a switch towards a more anabolic joint environment was observed. Further investigation of this timepoint and the pathways involved might elucidate the regenerative mechanisms behind KJD. The Translational Potential of this Article Further elucidation of the regenerative mechanisms behind KJD could improve the existing KJD treatment. Furthermore, these findings could provide input for the discovery or improvement of other joint regenerative treatment strategies

The stimulatory effect of notochordal cell-conditioned medium in a nucleus pulposus explant culture

Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their native environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect on extracellular matrix production. Methods: Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 μg/mL Link N. Furthermore, BM and NCCM samples were injected with 106 BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, glycosaminoglycan [GAG], hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining. Results: Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC-injected groups was not higher than in the other groups after 4 weeks of culture. Discussion: This study shows that, indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of an NCCM-based treatment for intervertebral disc regeneration

The effect of notochordal cell secreted factors on nucleus pulposus regeneration

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Stimulation of canine nucleus pulposus cells and bone marrow derived stromal cells with notochordal cell-secreted factors

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