Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture

<div><p>Aims</p><p>Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (<i>h</i>AMSCs) and bone-marrow-derived MSCs (<i>h</i>BMSCs) combined with bovine articular chondrocytes (<i>b</i>ACs) was compared.</p><p>Methods</p><p><i>h</i>AMSCs or <i>h</i>BMSCs were combined with <i>b</i>ACs in alginate and cultured <i>in vitro</i> or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between <i>b</i>ACs and <i>h</i>MSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted.</p><p>Results</p><p>The presence of <i>h</i>MSCs–either <i>h</i>AMSCs or <i>h</i>BMSCs—increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in <i>h</i>BMSC/<i>b</i>ACs. This effect was similar when <i>h</i>MSCs and <i>b</i>AC were combined in pellet culture, in alginate culture or when conditioned media of <i>h</i>MSCs were used on <i>b</i>AC. Species-specific gene-expression analyses demonstrated that <i>aggrecan</i> was expressed by <i>b</i>ACs only, indicating a predominantly trophic role for <i>h</i>MSCs. <i>Collagen-10</i>-gene expression of <i>b</i>ACs was not affected by <i>h</i>BMSCs, but slightly enhanced by <i>h</i>AMSCs. After <i>in-vivo</i> implantation, <i>h</i>AMSC/<i>b</i>ACs and <i>h</i>BMSC/<i>b</i>ACs had similar cartilage matrix production, both appeared stable and did not calcify.</p><p>Conclusions</p><p>This study demonstrates that replacing 80% of <i>b</i>ACs by either <i>h</i>AMSCs or <i>h</i>BMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by <i>h</i>MSCs.</p></div

Paracrine effect of <i>b</i>ACs on <i>h</i>AMSCs and <i>h</i>BMSCs.

<p><b>(A)</b> Schematic overview. In purple: <i>h</i>MSCs; in green: <i>b</i>ACs. <b>(B)</b> The DNA and GAG content of <i>h</i>AMSCs and <i>h</i>BMSCs in the presence of paracrine factors of <i>b</i>ACs via Transwell® system or <i>b</i>AC-conditioned medium. The DNA content after 3 weeks of culture was compared to the initial DNA content prior to cell-culture (dotted line). *, ** or *** indicates p-values smaller than 0.05, 0.01 or 0.001 respectively compared to the amount of DNA prior to cell culture. Data are shown as box-whisker plots of 6 samples of one experiment. For statistical evaluation, a Kruskal-Wallis followed by the Mann-Whitney-U test was use followed by a Bonferroni's post-hoc comparisons test. TW = Transwell; CM = Conditioned Medium; <i>h</i>AMSC = human Adipose-tissue-derived Mesenchymal Stem Cell; <i>h</i>BMSC = human Bone-marrow-derived Mesenchymal Stem Cell; <i>b</i>AC = bovine Articular Chondrocyte.</p

Gene-expression analysis, 5 weeks after <i>in vitro</i> culture.

<p>Data are shown as mean CT-values ± SD of housekeeping genes <b>(A)</b> and average relative gene-expression of chondrogenic genes <b>(B)</b>. nd = not detected (ct-value > 35.00); <i>hsGAPDH</i> = human-specific <i>GAPDH</i>; <i>bsGAPDH</i> = bovine-specific <i>GAPDH</i>; <i>hsACAN</i> = human-specific <i>ACAN</i>; <i>bsACAN</i> = bovine-specific <i>ACAN</i>; <i>hsCOL2A1</i> = human-specific COL2A1; <i>h</i>AMSC = human Adipose-tissue-derived Mesenchymal Stem Cell (<i>n = 3</i> experiments with 3 independent donors); <i>h</i>BMSC = human Bone-marrow-derived Mesenchymal Stem Cell (<i>n = 3</i> experiments with 3 independent donors); <i>b</i>AC = bovine Articular Chondrocyte (<i>n = 3</i> experiments with 3 pools of donors). Per experiment, 3 samples were used for analyses.</p

Cartilage matrix formation in constructs containing <i>h</i>MSCs and/or <i>b</i>ACs, 3 weeks after <i>in-vitro</i> culture.

<p><b>(A)</b> The DNA content of none of the constructs had changed compared to their initial DNA content prior to cell-culture (dotted line). Biochemical evaluation of the GAG <b>(B)</b> and collagen <b>(C)</b> content, 3 weeks after culture in alginate. The left graphs demonstrate the amount of matrix components per construct, whereas for the right graphs matrix production is normalized to the initially seeded primary ACs. A control condition—containing similar amounts of <i>b</i>ACs (0.8*10⁶ nc/ml) without supplementation of <i>h</i>MSCs—was evaluated to determine the additional effect of <i>h</i>MSCs (3.2*10⁶ nc/ml) on <i>b</i>ACs in co-cultures (dotted line). *, ** or *** indicates p-values smaller than 0.05, 0.01 or 0.001 respectively compared to the control condition. Data are shown as mean ± SD. For statistical evaluation, a mixed model was used followed by a Bonferroni's post-hoc comparisons test. <i>h</i>AMSC = human Adipose-tissue-derived Mesenchymal Stem Cell (<i>n = 3</i> experiments with 3 independent donors); <i>h</i>BMSC = human Bone-marrow-derived Mesenchymal Stem Cell (<i>n = 3</i> experiments with 3 independent donors); <i>b</i>AC = bovine Articular Chondrocyte (<i>n = 3</i> experiments with 3 pools of donors). Per experiment, 3 samples were used for analyses.</p

Macroscopic appearance and immunohistochemical analyses of constructs containing <i>h</i>MSCs and/or <i>b</i>ACs, 8 weeks after subcutaneous implantation in mice.

<p>Macroscopic appearance (top row) of cartilage constructs, as well as a collagen type II immunohistochemical staining (bottom rows), 8 weeks after subcutaneous implantation. <i>h</i>AMSC = human Adipose-tissue-derived Mesenchymal Stem Cell (<i>n = 3</i> experiments with 3 independent donors); <i>h</i>BMSC = human Bone-marrow-derived Mesenchymal Stem Cell (<i>n = 3</i> experiments with 3 independent donors); <i>b</i>AC = bovine Articular Chondrocyte (<i>n = 3</i> experiments with 3 pools of donors). Per experiment, 2 samples were used for analyses.</p

Reduced nasal IL-10 and enhanced TNFalpha responses during rhinovirus and RSV-induced upper respiratory tract infection in atopic and non-atopic infants

Rhinovirus and respiratory syncytial virus (RSV) are the most prevalent inducers of upper respiratory tract infections (URTI) in infants and may stimulate immune maturation. To estimate the amount of immune stimulation, nasal immune responses were examined during rhinovirus and RSV-induced URTI in infants. Nasal brush samples were taken from infants (2-26 months; 57% atopic family) with rhinovirus-induced URTI (N=20), with RSV-induced URTI (N=7), and with rhinovirus-induced rhinitis (N=11), from children with asymptomatic rhinovirus infection (N=7) and from eight non-infected children. Numbers of nasal brush cells positive for Th1-, Th2-, regulatory and proinflammatory cytokines were measured by immunohistochemistry or by measuring protein levels using a cytometric bead array analysis. During rhinovirus and RSV-induced URTI, fewer regulatory cytokine IL-10 positive cells were found compared to non-infected children. This fall was accompanied by an increase in levels of the Th1 cytokine TNFalpha. IL-10 responses were inversely related to TNFalpha responses. No enhanced responses were observed for IFNgamma, IL-12 and IL-18. Cytokine responses were comparable in children with rhinovirus-induced URTI and in children with rhinitis, while responses in asymptomatic rhinovirus-infected children were located between those for symptomatic and asymptomatic rhinovirus-infected children. Cytokine responses did not depend on the age of the child or atopy in the family. In conclusion, reduced nasal IL-10 responses during URTI in infants could facilitate the induction of a TNFalpha response. TNFalpha in turn could replace the immature production of IL-12, IL-18 and IFNgamma during URTI to induce an effective clearance of the viral infection and which could stimulate the maturation of Th1 cytokine production in infanc

Angiogenic Potential of Tissue Engineered Cartilage From Human Mesenchymal Stem Cells Is Modulated by Indian Hedgehog and Serpin E1

With rising demand for cartilage tissue repair and replacement, the differentiation of mesenchymal stem cells (BMSCs) into cartilage tissue forming cells provides a promising solution. Often, the BMSC-derived cartilage does not remain stable and continues maturing to bone through the process of endochondral ossification in vivo. Similar to the growth plate, invasion of blood vessels is an early hallmark of endochondral ossification and a necessary step for completion of ossification. This invasion originates from preexisting vessels that expand via angiogenesis, induced by secreted factors produced by the cartilage graft. In this study, we aimed to identify factors secreted by chondrogenically differentiated bone marrow-derived human BMSCs to modulate angiogenesis. The secretome of chondrogenic pellets at day 21 of the differentiation program was collected and tested for angiogenic capacity using in vitro endothelial migration and proliferation assays as well as the chick chorioallantoic membrane (CAM) assay. Taken together, these assays confirmed the pro-angiogenic potential of the secretome. Putative secreted angiogenic factors present in this medium were identified by comparative global transcriptome analysis between murine growth plate cartilage, human chondrogenic BMSC pellets and human neonatal articular cartilage. We then verified by PCR eight candidate angiogenesis modulating factors secreted by differentiated BMSCs. Among those, Serpin E1 and Indian Hedgehog (IHH) had a higher level of expression in BMSC-derived cartilage compared to articular chondrocyte derived cartilage. To understand the role of these factors in the pro-angiogenic secretome, we used neutralizing antibodies to functionally block them in the conditioned medium. Here, we observed a 1.4-fold increase of endothelial cell proliferation when blocking IHH and 1.5-fold by Serpin E1 blocking compared to unblocked control conditioned medium. Furthermore, endothelial migration was increased 1.9-fold by Serpin E1 blocking and 2.7-fold by IHH blocking. This suggests that the pro-angiogenic potential of chondrogenically differentiated BMSC secretome could be further augmented through inhibition of specific factors such as IHH and Serpin E1 identified as anti-angiogenic factors

Paracrine effect of <i>h</i>AMSCs and <i>h</i>BMSCs on <i>b</i>ACs.

<p><b>(A)</b> Schematic overview. In purple: <i>h</i>MSCs; in green: <i>b</i>ACs. <b>(B)</b> The DNA and GAG content of <i>b</i>ACs in the presence of paracrine factors of <i>h</i>MSCs via Transwell® system or <i>h</i>MSC-conditioned medium. The DNA content after 3 weeks of culture was compared to the initial DNA content prior to cell-culture (dotted line). <b>(C)</b> Co-culture in alginate constructs and pellet culture, allowing direct cell-cell contact. <b>(D)</b> Relative gene-expression analysis, 3 weeks after culture in alginate. *, ** or *** indicates p-values smaller than 0.05, 0.01 or 0.001 respectively compared to the amount of DNA prior to cell culture. # indicates significant differences from all conditions (<i>p</i><0.01). Data are shown as box-whisker plots of 6 samples of one experiment. For statistical evaluation, a Kruskal-Wallis followed by the Mann-Whitney-U test was use followed by a Bonferroni's post-hoc comparisons test. TW = Transwell; CM = Conditioned Medium; <i>h</i>AMSC = human Adipose-tissue-derived Mesenchymal Stem Cell; <i>h</i>BMSC = human Bone-marrow-derived Mesenchymal Stem Cell; <i>b</i>AC = bovine Articular Chondrocyte.</p

Cellular interaction.

<p>Cells were encapsulated in alginate beads separately and alginate and pellet co-cultures <b>(A, control conditions)</b>. Furthermore, <i>h</i>MSCs and <i>b</i>ACs were co-cultured in <b>(B)</b> a Transwell® system as well as in <b>(C)</b> medium conditioned by the other cell type, to further understand the complex cellular communication pathways between <i>h</i>MSCs and <i>b</i>ACs. In purple: <i>h</i>MSCs = human Mesenchymal Stem Cells; in green: <i>b</i>ACs = bovine Articular Chondrocytes.</p

Sequences of primers for qRT-PCR.

<p>Sequences of primers for qRT-PCR.</p