Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

The effect of bone allografts combined with bone marrow stromal cells on the healing of segmental bone defects in a sheep model

Background: The repair of large bone defects is a major orthopedic challenge because autologous bone grafts are not available in large amounts and because harvesting is often associated with donor-site morbidity. Considering that bone marrow stromal cells (BMSC) are responsible for the maintenance of bone turnover throughout life, we investigated bone repair at a site of a critically sized segmental defect in sheep tibia treated with BMSCs loaded onto allografts. The defect was created in the mid-portion of the tibial diaphysis of eight adult sheep, and the sheep were treated with ex-vivo expanded autologous BMSCs isolated from marrow aspirates and loaded onto cortical allografts (n = 4). The treated sheep were compared with control sheep that had been treated with cell-free allografts (n = 4) obtained from donors of the same breed as the receptor sheep. Results: The healing response was monitored by radiographs monthly and by computed tomography and histology at six, ten, fourteen, and eighteen weeks after surgery. For the cell-loaded allografts, union was established more rapidly at the interface between the host bone and the allograft, and the healing process was more conspicuous. Remodeling of the allograft was complete at 18 weeks in the cell-treated animals. Histologically, the marrow cavity was reestablished, with intertrabecular spaces being filled with adipose marrow and with evidence of focal hematopoiesis. Conclusions: Allografts cellularized with AOCs (allografts of osteoprogenitor cells) can generate great clinical outcomes to noncellularized allografts to consolidate, reshape, structurally and morphologically reconstruct bone and bone marrow in a relatively short period of time. These features make this strategy very attractive for clinical use in orthopedic bioengineering

Mesenchymal stromal cell: new applications for regenerative medicine

In the last decades mesenchymal stromal cells (MSC), intriguing for their multilineage plasticity and their proliferation activity in vitro, have been intensively studied for innovative therapeutic applications. In the first project, a new method to expand in vitro adipose derived-MSC (ASC) while maintaining their progenitor properties have been investigated. ASC are cultured in the same flask for 28 days in order to allow cell-extracellular matrix and cell-cell interactions and to mimic in vivo niche. ASC cultured with this method (Unpass cells) were compared with ASC cultured under classic condition (Pass cells). Unpass and Pass cells were characterized in terms of clonogenicity, proliferation, stemness gene expression, differentiation in vitro and in vivo and results obtained showed that Unpass cells preserve their stemness and phenotypic properties suggesting a fundamental role of the niche in the maintenance of ASC progenitor features. Our data suggests alternative culture conditions for the expansion of ASC ex vivo which could increase the performance of ASC in regenerative applications. In vivo MSC tracking is essential in order to assess their homing and migration. Super-paramagnetic iron oxide nanoparticles (SPION) have been used to track MSC in vivo due to their biocompatibility and traceability by MRI. In the second project a new generation of magnetic nanoparticles (MNP) used to label MSC were tested. These MNP have been functionalized with hyperbranched poly(epsilon-lysine)dendrons (G3CB) in order to interact with membrane glycocalix of the cells avoiding their internalization and preventing any cytotoxic effects. In literature it is reported that labeling of MSC with SPION takes long time of incubation. In our experiments after 15min of incubation with G3CB-MNP more then 80% of MSC were labeled. The data obtained from cytotoxic, proliferation and differentiation assay showed that labeling does not affect MSC properties suggesting a potential application of G3CB nano-particles in regenerative medicine.

Mesenchymal Stromal Cells (MSCs) and induced Plutipotent Stem Cells (iPSCs) in Domestic Animals: Characterization and Differentiation Potential

Derivation of stem cell lines from domesticated animals has been of great interest as it benefits translational medicine, clinical applications to improve human and animal health and biotechnology. The main types of stem cells studied are Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and Mesenchymal Stem/Stromal Cells (MSCs). This thesis had two main aims: (I) The isolation of bovine MSCs from amniotic fluid (AF) at different trimesters of pregnancy and their characterization to study pluripotency markers expression. Stemness markers were studied also in MSCs isolated from equine AF, Wharton’s jelly (WJ) and umbilical cord blood (UCB) as continuation of the characterization of these cells previously performed by our research group; (II) The establishment and characterization of iPSCs lines in two attractive large animal models for biomedical and biotechnology research such as the bovine and the swine, and the differentiation into the myogenic lineage of porcine iPSCs. It was observed that foetal tissues in domestic animals such as the bovine and the horse represent a source of MSCs able to differentiate into the mesodermal lineage but they do not proliferate indefinitely and they lack the expression of many pluripotency markers, making them an interesting source of cells for regenerative medicine, but not the best candidate to elucidate pluripotency networks. The protocol used to induce pluripotency in bovine fibroblasts did not work, as well as the chemical induction of pluripotency in porcine fibroblasts, while the reprogramming protocol used for porcine iPSCs was successful and the line generated was amenable to being differentiated into the myogenic lineage, demonstrating that they could be addressed into a desired lineage by genetic modification and appropriated culture conditions. Only a few cell types have been differentiated from domestic animal iPSCs to date, so the development of a reliable directed-differentiation protocol represents a very important result.

Contribution of vascular resident mesenchymal stromal cells to abdominal aortic aneurysm pathogenesis: increased MMP-9 expression and ineffective immunomodulation

Background. Ageing and inflammation are critical for the occurrence of aortic diseases. Extensive inflammatory infiltrate and excessive ECM proteloysis, mediated by MMPs, are typical features of abdominal aortic aneurysm (AAA). Mesenchymal Stromal Cells (MSCs) have been detected within the vascular wall and represent attractive candidates for regenerative medicine, in virtue of mesodermal lineage differentiation and immunomodulatory activity. Meanwhile, many works have underlined an impaired MSC behaviour under pathological conditions. This study was aimed to define a potential role of vascular MSCs to AAA development. Methods. Aortic tissues were collected from AAA patients and healthy donors. Our analysis was organized on three levels: 1) histology of AAA wall; 2) detection of MSCs and evaluation of MMP-9 expression on AAA tissue; 3) MSC isolation from AAA wall and characterization for mesenchymal/stemness markers, MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN. AAA-MSCs were tested for immunomodulation, when cultured together with activated peripheral blood mononuclear cells (PBMCs). In addition, a co-colture of both healthy and AAA MSCs was assessed and afterwards MMP-2/9 mRNA levels were analyzed. Results. AAA-MSCs showed basic mesenchymal properties: fibroblastic shape, MSC antigens, stemness genes. MMP-9 mRNA, protein and enzymatic activity were significantly increased in AAA-MSCs. Moreover, AAA-MSCs displayed a weak immunosuppressive activity, as shown by PBMC ongoing along cell cycle. MMP-9 was shown to be modulated at the transcriptional level through the direct contact as well as the paracrine action of healthy MSCs. Discussion. Vascular injury did not affect the MSC basic phenotype, but altered their function, a increased MMP-9 expression and ineffective immunmodulation. These data suggest that vascular MSCs can contribute to aortic disease. In this view, the study of key processes to restore MSC immunomodulation could be relevant to find a pharmacological approach for monitoring the aneurysm progression.

Biomechanical model of human cornea based on stromal microstructure

The optical characteristics of the human cornea depends on the mechanical balance between the intra-ocular pressure and intrinsic tissue stiffness. A wide range of ophthalmic surgical procedures alter corneal biomechanics to induce local or global curvature changes for the correction of visual acuity. Due to the large number of surgical interventions performed every day, a deeper understanding of corneal biomechanics is needed to improve the safety of these procedures and medical devices. The aim of this study is to propose a biomechanical model of the human cornea, based on stromal microstructure. The constitutive mechanical law includes collagen fiber distribution based on X-ray scattering analysis, collagen cross-linking, and fiber uncrimping. Our results showed that the proposed model reproduced inflation and extensiometry experimental data [Elsheikh et al., Curr. Eye Res., 2007; Elsheikh et al., Exp. Eye Res., 2008] successfully. The mechanical properties obtained for different age groups demonstrated an increase in collagen cross-linking for older specimens. In future work such a model could be used to simulate non-symmetric interventions, and provide better surgical planning.

Numerical model of the human cornea based on stromal microstructure: identification of mechanical properties for two age groups

The optical quality of the human eye mainly depends on the refractive performance of the cornea. The shape of the cornea is a mechanical balance between intraocular pressure and tissue intrinsic stiffness. Several surgical procedures in ophthalmology alter the biomechanics of the cornea to provoke local or global curvature changes for vision correction. Legitimated by the large number of surgical interventions performed every day, the demand for a deeper understanding of corneal biomechanics is rising to improve the safety of procedures and medical devices. The aim of our work is to propose a numerical model of corneal biomechanics, based on the stromal microstructure. Our novel anisotropic constitutive material law features a probabilistic weighting approach to model collagen fiber distribution as observed on human cornea by Xray scattering analysis (Aghamohammadzadeh et. al., Structure, February 2004). Furthermore, collagen cross-linking was explicitly included in the strain energy function. Results showed that the proposed model is able to successfully reproduce both inflation and extensiometry experimental data (Elsheikh et. al., Curr Eye Res, 2007; Elsheikh et. al., Exp Eye Res, May 2008). In addition, the mechanical properties calculated for patients of different age groups (Group A: 65-79 years; Group B: 80-95 years) demonstrate an increased collagen cross-linking, and a decrease in collagen fiber elasticity from younger to older specimen. These findings correspond to what is known about maturing fibrous biological tissue. Since the presented model can handle different loading situations and includes the anisotropic distribution of collagen fibers, it has the potential to simulate clinical procedures involving nonsymmetrical tissue interventions. In the future, such mechanical model can be used to improve surgical planning and the design of next generation ophthalmic devices.

Differential response of Human Bone Marrow Stromal Cells to either TGF-β1 or to rhGDF-5

Cell therapy along with growth factor injection is currently widely investigated to restore the intervertebral disc. However, there is increasing evidence that transplanted unconditioned bone marrow-derived stromal cells (BMSCs) cannot thrive in the intervertebral disc "niche". Moreover, uncertainty exists with respect to the cell phenotype that would be suitable to inject. The intervertebral disc cell phenotype only recently has been started to be characterised using transcriptomics profiling. Recent findings suggest that cytokeratin 19 (KRT-19) could be used as a potential candidate marker for the intervertebral disc, or more specifically the nucleus pulposus cell (NPC) phenotype. We present in vitro cell culture data using alginate bead culture of primary human BMSCs exposed to the standard chondrogenic stimulus, transforming growth factor beta-1 (TGF-β), the growth and differentiation factor 5 and/or bovine NPCs to induce a potential "discogenic" pathway. Chondrogenic induction via TGF-β pathway provoked down-regulation of KRT-19 gene expression in four out of five donors after 18 days of culture, whereas KRT-19 expression remained unchanged in the "discogenic" groups. In addition, the ratio of aggrecan/collagen II gene expression showed a remarkable difference (of at least 3 magnitudes) between the chondrogenic stimulus (low ratio) and the discogenic stimulus (high ratio). Therefore, KRT-19 and aggrecan/collagen II ratio may be potential markers to distinguish chondrogenic from "discogenic" differentiation.

Dose-response effect of interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, and interferon-y on the in vitro production of epithelial neutrophil activating peptide-78 (ENA-78), IL-8, and IL-6 by human endometrial stromal cells

The production of epithelial neutrophil activating peptide-78 (NA-78) and the interleukins IL-8 and IL-6 by endometrial stromal cells is stimulated by pro-inflammatory interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α). IL-8 is suggested to play a role in the pathogenesis of endometriosis, and in these women the peritoneal fluid concentrations of ENA-78 and IL-8 are increased. TNF-α has been tested together with interferon-γ because of their cooperative stimulation of IL-6. The release of IL-8, however, is inhibited with increasing interferon levels. The aim of the study was the analysis of the production of ENA-78, IL-6 and IL-8 by cultured human endometrial stromal cells in the presence of varying concentrations of IL-1β, TNF-α, and interferon-γ.