MESENCHYMAL STEM CELL-MEDIATED ENDOCHONDRAL OSSIFICATION UTILISING MICROPELLETS AND BRIEF CHONDROGENIC PRIMING

With limited autologous and donor bone graft availability, there is an increasing need for alternative graft substitutes. We have previously shown that chondrogenically priming mesenchymal stem cell (MSC) pellets for 28 d in vitro will reproducibly result in endochondral bone formation after in vivo implantation. However, pellet priming time for clinical applications is quite extensive. A micropellet (\xce\xbcpellet)-fibrin construct was developed and coupled, with a shorter priming period, determined by an in vitro time course experiment. In vitro data showed expression of chondrogenic genes and matrix production after 7 d of chondrogenic priming, indicating that briefer priming could possibly be used to induce bone formation in vivo. 7 and 28 d primed pellet, pellet-fibrin and \xce\xbcpellet-fibrin constructs were cultured for in vitro analysis and implanted subcutaneously for 8 weeks into nude mice. \xce\xbcpellet-fibrin constructs, cultured in vitro for 7 or 28 d, produced comparable bone to standard pellets in vivo. MSC-mediated bone formation was achieved following only 7 d of in vitro priming. Bone formation in vivo appeared to be influenced by overall matrix production pre-implantation. Given this short priming time and the injectable nature of the \xce\xbcpellet-fibrin constructs, this approach might be further developed as an injectable bone substitute, leading to a minimally-invasive treatment option, which would allow for tailored filling of bone defects

Bio-inspired polymeric iron-doped hydroxyapatite microspheres as a tunable carrier of rhBMP-2

Hybrid superparamagnetic microspheres with bone-like composition, previously developed by a bio-inspired assembling/mineralization process, are evaluated for their ability to uptake and deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) in therapeutically-relevant doses along with prolonged release pro- files. The comparison with hybrid non-magnetic and with non-mineralized microspheres highlights the role of nanocrystalline, nanosize mineral phases when they exhibit surface charged groups enabling the chemical linking with the growth factor and thus moderating the release kinetics. All the microspheres show excellent osteogenic ability with human mesenchymal stem cells whereas the hybrid mineralized ones show a slow and sustained release of rhBMP-2 along 14 days of soaking into cell culture medium with substantially bioactive effect, as reported by assay with C2C12 BRE-Luc cell line. It is also shown that the release extent can be modulated by the application of pulsed electromagnetic field, thus showing the potential of remote controlling the bioactivity of the new micro-devices which is promising for future application of hybrid biomimetic mi- crospheres in precisely designed and personalized therapies.info:eu-repo/semantics/publishedVersio

The response of human macrophages to 3D printed titanium antibacterial implants does not affect the osteogenic differentiation of hMSCs

Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.</p

Chondrogenically Primed Human Mesenchymal Stem Cells Persist and Undergo Early Stages of Endochondral Ossification in an Immunocompetent Xenogeneic Model

Tissue engineering approaches using progenitor cells such as mesenchymal stromal cells (MSCs) represent a promising strategy to regenerate bone. Previous work has demonstrated the potential of chondrogenically primed human MSCs to recapitulate the process of endochondral ossification and form mature bone in vivo, using immunodeficient xenogeneic models. To further the translation of such MSC-based approaches, additional investigation is required to understand the impact of interactions between human MSC constructs and host immune cells upon the success of MSC-mediated bone formation. Although human MSCs are considered hypoimmunogenic, the potential of chondrogenically primed human MSCs to induce immunogenic responses in vivo, as well as the efficacy of MSC-mediated ectopic bone formation in the presence of fully competent immune system, requires further elucidation. Therefore, the aim of this study was to investigate the capacity of chondrogenically primed human MSC constructs to persist and undergo the process of endochondral ossification in an immune competent xenogeneic model. Chondrogenically differentiated human MSC pellets were subcutaneously implanted to wild-type BALB/c mice and retrieved at 2 and 12 weeks post-implantation. The percentages of CD4(+) and CD8(+) T cells, B cells, and classical/non-classical monocyte subsets were not altered in the peripheral blood of mice that received chondrogenic MSC constructs compared to sham-operated controls at 2 weeks post-surgery. However, MSC-implanted mice had significantly higher levels of serum total IgG compared to sham-operated mice at this timepoint. Flow cytometric analysis of retrieved MSC constructs identified the presence of T cells and macrophages at 2 and 12 weeks post-implantation, with low levels of immune cell infiltration to implanted MSC constructs detected by CD45 and CD3 immunohistochemical staining. Despite the presence of immune cells in the tissue, MSC constructs persisted in vivo and were not degraded/resorbed. Furthermore, constructs became mineralised, with longitudinal micro-computed tomography imaging revealing an increase in mineralised tissue volume from 4 weeks post-implantation until the experimental endpoint at 12 weeks. These findings indicate that chondrogenically differentiated human MSC pellets can persist and undergo early stages of endochondral ossification following subcutaneous implantation in an immunocompetent xenogeneic model. This scaffold-free model may be further extrapolated to provide mechanistic insight to osteoimmunological processes regulating bone regeneration and homeostasis

Endoplasmic Reticulum Stress, Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis

Background:Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.Methods:Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.Results:Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.Conclusions:XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury

Cytological Studies on the Anterior Pituitary in the Goat (IV) : Changes of the anterior pituitary cells following castration

textabstractBackground: Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha'-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha'-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2-/-) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer. Methods: Muc2-/- mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed. Results: Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2-/- mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis. Conclusions: This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2-/- mice by inducing an immunosuppressive Treg cell response

Pediatric mesenchymal stem cells exhibit immunomodulatory properties toward allogeneic T and B cells under inflammatory conditions

Mesenchymal stem cells from pediatric patients (pMSCs) are an attractive cell source in regenerative medicine, due to their higher proliferation rates and better differentiation abilities compared to adult MSCs (aMSCs). We have previously characterized the immunomodulatory abilities of pMSCs on T cells under co-culture. It has also been reported that aMSCs can inhibit B cell proliferation and maturation under inflammatory conditions. In this study, we therefore aimed to clarify the immunomodulatory effect of pMSCs toward T and B cells in an inflammatory microenvironment. Bone marrow derived pMSCs were primed to simulate inflammatory conditions by exposure with 50 ng/mL of IFN-γ for 3 days. To analyze the interaction between pMSCs and T cells, CD3/CD28 stimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with primed or unprimed pMSCs. To investigate B cell responses, quiescent B cells obtained from spleens by CD43 negative selection were stimulated with anti-IgM, anti-CD40, IL-2, and co-cultured with either IFN-γ primed or unprimed pMSC. pMSC phenotype, B and T cell proliferation, and B cell functionality were analyzed. Gene expression of indoleamine 2,3-dioxygenease (IDO), as well as the expression of HLA-ABC, HLA-DR and the co-stimulatory molecules CD80 and CD86 was upregulated on pMSCs upon IFN-γ priming. IFN-γ did not alter the immunomodulatory abilities of pMSCs upon CD4+ nor CD8+ stimulated T cells compared to unprimed pMSCs. IFN-γ primed pMSCs but not unprimed pMSCs strongly inhibited naïve (CD19+CD27-), memory (CD19+CD27+), and total B cell proliferation. Antibody-producing plasmablast (CD19+CD27highCD38high) formation and IgG production were also significantly inhibited by IFN-γ primed pMSCs compared to unprimed pMSCs. Collectively, these results show that pMSCs have immunomodulatory effects upon the adaptive immune response which can be potentiated by inflammatory stimuli. This knowledge is useful in regenerative medicine and allogeneic transplantation applications toward tailoring pMSCs function to best modulate the immune response for a successful implant engraftment and avoidance of a strong immune reaction

Mucin Muc2 Deficiency and Weaning Influences the Expression of the Innate Defense Genes Reg3β, Reg3γ and Angiogenin-4

Background Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2-/-) on the expression of regenerating islet-derived protein 3 beta (Reg3ß), regenerating islet-derived protein 3 gamma (Reg3¿), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. Methods Intestinal tissues of Muc2-/- and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3ß, Reg3¿, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. Results Reg3ß and Reg3¿ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3ß, Reg3¿, and Ang4 differed between WT and Muc2-/- mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3ß and Reg3¿ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2-/- mice at P28, where and when expression levels of Reg3ß, Reg3¿, and Ang4 were the lowest. Conclusions Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3ß and Reg3¿ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2-/- mice. The inverse correlation between colitis development and Reg3ß, Reg3¿, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammatio

Collagen type X is essential for successful mesenchymal stem cell-mediated cartilage formation and subsequent endochondral ossification

n tissue engineering, endochondral ossification (EO) is often replicated by chondrogenically differentiating mesenchymal stromal cells (MSCs) in vitro and achieving bone formation through in vivo implantation. The resulting marrow-containing bone constructs are promising as a treatment for bone defects. However, limited bone formation capacity has prevented them from reaching their full potential. This is further complicated since it is not fully understood how this bone formation is achieved. Acellular grafts derived from chondrogenically differentiated MSCs can initiate bone formation; however, which component within these decellularised matrices contribute to bone formation has yet to be determined. Collagen type X (COLX), a hypertrophy-associated collagen found within these constructs, is involved in matrix organisation, calcium binding and matrix vesicle compartmentalisation. However, the importance of COLX during tissue-engineered chondrogenesis and subsequent bone formation is unknown. The present study investigated the importance of COLX by shRNA-mediated gene silencing in primary MSCs. A significant knock-down of COLX disrupted the production of extracellular matrix key components and the secretion profile of chondrogenically differentiated MSCs. Following in vivo implantation, disrupted bone formation in knock-down constructs was observed. The importance of COLX was confirmed during both chondrogenic differentiation and subsequent EO in this tissue engineered setting

Isolating Pediatric Mesenchymal Stem Cells with Enhanced Expansion and Differentiation Capabilities

Mesenchymal stem cells/marrow stromal cells (MSCs) are attractive for applications ranging from research and development to use in clinical therapeutics. However, the most commonly studied MSCs, adult bone marrow MSCs (A-MSCs), are limited by significant donor variation resulting in inconsistent expansion rates and multilineage differentiation capabilities. We have recently obtained permission to isolate pediatric MSCs (P-MSCs) from surplus iliac crest bone chips. Here, we developed a simple and easily replicable isolation protocol yielding P-MSCs, which adhere to MSC defining guidelines. After confirming immunophenotypic marker expression, we compared expansion rates, senescence, morphology, and trilineage differentiation of P-MSCs to A-MSCs for multiple donors. We found P-MSCs have faster in vitro replication, consistently show significantly lower senescence, and are capable of more reproducible multilineage differentiation than A-MSCs. We, therefore, believe P-MSCs are a promising candidate for use in research applications and potentially as part of an allogeneic therapeutic treatment