Patricio C. Smith

The influence of platelet-derived products on angiogenesis and tissue repair: a concise update

Platelet degranulation allows the release of a large amount of soluble mediators, is an essential step for wound healing initiation, and stimulates clotting, and angiogenesis. The latter process is one of the most critical biological events observed during tissue repair, increasing the growth of blood vessels in the maturing wound. Angiogenesis requires the action of a variety of growth factors that act in an appropriate physiological ratio to assure functional blood vessel restoration. Platelets release main regulators of angiogenesis: Vascular Endothelial Growth Factors (VEGFs), basic fibroblast growth factor (FGF-2), and Platelet derived growth factors (PDGFs), among others. In order to stimulate tissue repair, platelet derived fractions have been used as an autologous source of growth factors and biomolecules, namely Platelet Rich Plasma (PRP), Platelet Poor Plasma (PPP), and Platelet Rich Fibrin (PRF). The continuous release of these growth factors has been proposed to promote angiogenesis both in vitro and in vivo. Considering the existence of clinical trials currently evaluating the efficacy of autologous PRP, the present review analyses fundamental questions regarding the putative role of platelet derived fractions as regulators of angiogenesis and evaluates the possible clinical implications of these formulations.

NOX4-dependent ROS production by stromal mammary cells modulates epithelial MCF-7 cell migration

Background:The influence of the stromal microenvironment on the progression of epithelial cancers has been demonstrated. Unravelling the mechanisms by which stromal cells affect epithelial behaviour will contribute in understanding cellular malignancy. It has been proposed that redox environment has a role in the acquisition of malignancy. In this work, we studied the influence of epithelial cells on the stromal redox status and the consequence of this phenomenon on MCF-7 cell motility.Methods:We analysed in a co-culture system, the effect of RMF-EG mammary stromal cells on the migratory capacity of MCF-7 cell line. To test whether the NOX-dependent stromal redox environment influences the epithelial migratory behaviour, we knocked down the expression of NOX4 using siRNA strategy. The effect of TGF-β1 on NOX4 expression and activity was analysed by qPCR, and intracellular ROS production was measured by a fluorescent method.Results:Migration of MCF-7 breast epithelial cells was stimulated when co-cultured with RMF-EG cells. This effect depends on stromal NOX4 expression that, in turn, is enhanced by epithelial soluble factors. Pre-treatment of stromal cells with TGF-β1 enhanced this migratory stimulus by elevating NOX4 expression and intracellular ROS production. TGF-β1 seems to be a major component of the epithelial soluble factors that stimulate NOX4 expression.Conclusions:Our results have identified that an increased stromal oxidative status, mainly provided by an elevated NOX4 expression, is a permissive element in the acquisition of epithelial migratory properties. The capacity of stromal cells to modify their intracellular ROS production, and accordingly, to increase epithelial motility, seems to depend on epithelial soluble factors among which TGF-β1 have a decisive role.

RAC1 activity and intracellular ROS modulate the migratory potential of MCF-7 cells through a NADPH oxidase and NFκB-dependent mechanism

In the present study, we demonstrated that changes in Rac1 activity associated with the production of intracellular ROS modulate the migratory properties in MCF-7 and T47D human mammary cell lines. We also described that the NFkappaB pathway exerts a downstream control on the expression of the ROS-dependent cellular migratory potential. These results emphasize the importance of redox balance in the acquisition of malignancy and support previous data sustaining that an oxidative environment predisposes cells to activate signal-transduction pathways actively involved in cellular oncogenesis. Our data also provides evidence that NADPH oxidase could constitute the main source of intracellular ROS in response to changes in Rac1 activity. We suggest that Rac1 plays a role in cellular migration not only limited to its known function in reorganization of the actin cytoskeleton, but also as part of the intracellular machinery that controls the redox balance.

Soluble factors derived from tumor mammary cell lines induce a stromal mammary adipose reversion in human and mice adipose cells. Possible role of TGF-β1 and TNF-α

In carcinomas such as those of breast, pancreas, stomach, and colon, cancer cells support the expansion of molecular and cellular stroma in a phenomenon termed desmoplasia, which is characterized by a strong fibrotic response. In the case of breast tissue, in which stroma is mainly a fatty tissue, this response presumably occurs at the expense of the adipose cells, the most abundant stromal phenotype, generating a tumoral fibrous structure rich in fibroblast-like cells. In this study, we aimed to determine the cellular mechanisms by which factors present in the media conditioned by MDA-MB-231 and MCF-7 human breast cancer cell lines induce a reversion of adipose cells to a fibroblastic phenotype. We demonstrated that soluble factors generated by these cell lines stimulated the reversion of mammary adipose phenotype evaluated as intracellular lipid content and expression of C/EBPα and PPARγ. We also demonstrated that exogenous TGF-β1 and TNF-α exerts a similar function. The participation of both growth factors, components of media conditioned by tumoral mammary cells, on the expression and nuclear translocation of C/EBPα and PPARγ was tested in 3T3-L1 cells by interfering with the inhibitory effects of media with agents that block the TGF-β1 and TNF-α activity. These results allow us to postulate that TGF-β1 and TNF-α present in this media are in part responsible for this phenotypic reversion.

Defective Wound-healing in Aging Gingival Tissue

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process.

Effects of Chitosan Particles in Periodontal Pathogens and Gingival Fibroblasts

Chitosan is a naturally derived polymer with antimicrobial and anti-inflammatory properties. However, studies evaluating the role of chitosan in the control of periodontal pathogens and the responses of fibroblasts to inflammatory stimuli are lacking. In the present study, we analyzed whether chitosan particles may inhibit the growth of periodontal pathogens and modulate the inflammatory response in human gingival fibroblasts. Chitosan particles were generated through ionic gelation. They inhibited the growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans at 5 mg/mL. Conversely, IL-1β strongly stimulated PGE2 protein levels in gingival fibroblasts, and chitosan inhibited this response at 50 µg/mL. IL-1β–stimulated PGE2 production was dependent on the JNK pathway, and chitosan strongly inhibited this response. IL-1β stimulated NF-κB activation, another signaling pathway involved in PGE2 production. However, chitosan particles were unable to modify NF-κB signaling. The present study shows that chitosan exerts a predominantly anti-inflammatory activity by modulating PGE2 levels through the JNK pathway, which may be useful in the prevention or treatment of periodontal inflammation.

Synthesis of nanostructured porous silica coatings on titanium and their cell adhesive and osteogenic differentiation properties

Nanostructured porous silica coatings were synthesized on titanium by the combined sol-gel and evaporation-induced self-assembly process. The silica-coating structures were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and nitrogen sorptometry. The effect of the nanoporous surface on apatite formation in simulated body fluid, protein adsorption, osteoblast cell adhesion behavior, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) is reported. Silica coatings with highly ordered sub-10 nm porosity accelerate early osteoblast adhesive response, a favorable cell response that is attributed to an indirect effect due to the high protein adsorption observed on the large-specific surface area of the nanoporous coating but is also probably due to direct mechanical stimulus from the nanostructured topography. The nanoporous silica coatings, particularly those doped with calcium and phosphate, also promote the osteogenic differentiation of hBMSCs with spontaneous mineral nodule formation in basal conditions. The bioactive surface properties exhibited by the nanostructured porous silica coatings make these materials a promising alternative to improve the osseointegration properties of titanium dental implants and could have future impact on the nanoscale design of implant surfaces.

Effects of platelet‐rich and ‐poor plasma on the reparative response of gingival fibroblasts

OBJECTIVES Although platelet-rich plasma (PRP) has been proposed as a therapeutic tool to enhance wound repair, the cellular and molecular mechanisms stimulated by this agent are still not completely understood. The present study was designed to characterize the effects of PRP and platelet-poor plasma (PPP) supernatants on cell responses involved in gingival tissue repair. METHODS We studied the response of human gingival fibroblasts (HGF) to PRP and PPP fractions on: matrix contraction, cell migration, myofibroblastic differentiation, production of matrix components and proteolytic enzymes. PRP and PPP were obtained from donors using a commercial kit. Matrix contraction was evaluated by means of collagen lattices in the presence of matrix metalloproteinase (MMP) and actin polymerization inhibitors. The production of matrix molecules and proteinases was assessed through Western-blot. RhoA activity was evaluated through a pull-down assay. Actin distribution and focal adhesions were assessed through immunofluorescence. Transforming growth factor-beta (TGF-β) was quantified through ELISA. RESULTS Both PRP and PPP stimulated human gingival fibroblasts-populated collagen gel contraction and Ilomastat and cytochalasin D inhibited this response. PRP and PPP also stimulated MT1-MMP and TIMP-2 production, RhoA activation and actin cytoskeleton remodeling, cell migration/invasion and myofibroblastic differentiation. TGF-β1 was found at significantly higher concentrations in PRP than in PPP. CONCLUSIONS Both PRP and PPP promote wound tissue remodeling and contraction through the stimulation of actin remodeling, the activity of MMPs, promotion of cell migration, and myofibroblastic differentiation. The similar biological responses induced by PRP and PPP suggest that both platelet-derived fractions may exert a positive effect on gingival repair.

Tumor Necrosis Factor-α Inhibits Transforming Growth Factor-β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

BACKGROUND Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) modulate myofibroblastic differentiation and the production of ECM components. METHODS Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-β1 and TNF-α. Protein levels of α-smooth muscle actin (α-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α-SMA and actin fibers were identified by immunofluorescence. TGF-β1, TNF-α, and α-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-β1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs. RESULTS TGF-β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α-SMA and ED-A-FN. Moreover, TGF-β1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-α and TGF-β1 significantly reduced the expression of all the above-mentioned proteins. TNF-α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter. CONCLUSIONS TNF-α inhibits several cell responses induced by TGF-β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.

Preparation and bioactive properties of novel bone-repair bionanocomposites based on hydroxyapatite and bioactive glass nanoparticles †

Bionanocomposites based on ceramic nanoparticles and a biodegradable porous matrix represent a promising strategy for bone repair applications. The preparation and bioactive properties of bionanocomposites based on hydroxyapatite (nHA) and bioactive glass (nBG) nanoparticles were presented. nHA and nBG were synthesized with nanometric particle size using sol-gel/precipitation methods. Composite scaffolds were prepared by incorporating nHA and nBG into a porous alginate (ALG) matrix at different particle loads. The ability of the bionanocomposites to induce the crystallization of the apatite phase from simulated body fluid (SBF) was systematically evaluated using X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis, and Fourier transform infrared spectroscopy. Both nHA/ALG and nBG/ALG composites were shown to notably accelerate the process of crystallization and growth of the apatite phase on the scaffold surfaces. For short immersion times in SBF, nBG (25%)-based nanocomposites induced a higher degree of apatite crystallization than nHA (25%)-based nanocomposites, probably due to the more reactive nature of the BG particles. Through a reinforcement effect, the nanoparticles also improve the mechanical properties and stability in SBF of the polymer scaffold matrix. In addition, in vitro biocompatibility tests demonstrated that osteoblast cells are viable and adhere well on the surface of the bionanocomposites. These results indicate that nHA- and nBG-based bionanocomposites present potential properties for bone repair applications, particularly oriented to accelerate the bone mineralization process.