Synthesis of a Novel Electrospun Polycaprolactone Scaffold Functionalized with Ibuprofen for Periodontal Regeneration: An In Vitro andIn Vivo Study.

Ibuprofen (IBU) has been shown to improve periodontal treatment outcomes. The aim of this study was to develop a new anti-inflammatory scaffold by functionalizing an electrospun nanofibrous poly-ε-caprolactone membrane with IBU (IBU-PCL) and to evaluate its impact on periodontal inflammation, wound healing and regeneration in vitro and in vivo. IBU-PCL was synthesized through electrospinning. The effects of IBU-PCL on the proliferation and migration of epithelial cells (EC) and fibroblasts (FB) exposed to Porphyromonas gingivlais lipopolysaccharide (Pg-LPS) were evaluated through the AlamarBlue test and scratch assay, respectively. Anti-inflammatory and remodeling properties were investigated through Real time qPCR. Finally, the in vivo efficacy of the IBU-PCL membrane was assessed in an experimental periodontitis mouse model through histomorphometric analysis. The results showed that the anti-inflammatory effects of IBU on gingival cells were effectively amplified using the functionalized membrane. IBU-PCL reduced the proliferation and migration of cells challenged by Pg-LPS, as well as the expression of fibronectin-1, collagen-IV, integrin α3ß1 and laminin-5. In vivo, the membranes significantly improved the clinical attachment and IBU-PCL also reduced inflammation-induced bone destruction. These data showed that the IBU-PCL membrane could efficiently and differentially control inflammatory and migratory gingival cell responses and potentially promote periodontal regeneration.

Porphyromonas gingivalis Differentially Modulates Cell Death Profile in Ox-LDL and TNF-α Pre-Treated Endothelial Cells.

OBJECTIVE: Clinical studies demonstrated a potential link between atherosclerosis and periodontitis. Porphyromonas gingivalis (Pg), one of the main periodontal pathogen, has been associated to atheromatous plaque worsening. However, synergism between infection and other endothelial stressors such as oxidized-LDL or TNF-α especially on endothelial cell (EC) death has not been investigated. This study aims to assess the role of Pg on EC death in an inflammatory context and to determine potential molecular pathways involved. METHODS: Human umbilical vein ECs (HUVECs) were infected with Pg (MOI 100) or stimulated by its lipopolysaccharide (Pg-LPS) (1µg/ml) for 24 to 48 hours. Cell viability was measured with AlamarBlue test, type of cell death induced was assessed using Annexin V/propidium iodide staining. mRNA expression regarding caspase-1, -3, -9, Bcl-2, Bax-1 and Apaf-1 has been evaluated with RT-qPCR. Caspases enzymatic activity and concentration of APAF-1 protein were evaluated to confirm mRNA results. RESULTS: Pg infection and Pg-LPS stimulation induced EC death. A cumulative effect has been observed in Ox-LDL pre-treated ECs infected or stimulated. This effect was not observed in TNF-α pre-treated cells. Pg infection promotes EC necrosis, however, in infected Ox-LDL pre-treated ECs, apoptosis was promoted. This effect was not observed in TNF-α pre-treated cells highlighting specificity of molecular pathways activated. Regarding mRNA expression, Pg increased expression of pro-apoptotic genes including caspases-1,-3,-9, Bax-1 and decreased expression of anti-apoptotic Bcl-2. In Ox-LDL pre-treated ECs, Pg increased significantly the expression of Apaf-1. These results were confirmed at the protein level. CONCLUSION: This study contributes to demonstrate that Pg and its Pg-LPS could exacerbate Ox-LDL and TNF-α induced endothelial injury through increase of EC death. Interestingly, molecular pathways are differentially modulated by the infection in function of the pre-stimulation.

Active implant combining human stem cell microtissues and growth factors for bone-regenerative nanomedicine.

AIMS: Mesenchymal stem cells (MSCs) from adult bone marrow provide an exciting and promising stem cell population for the repair of bone in skeletal diseases. Here, we describe a new generation of collagen nanofiber implant functionalized with growth factor BMP-7 nanoreservoirs and equipped with human MSC microtissues (MTs) for regenerative nanomedicine. MATERIALS & METHODS: By using a 3D nanofibrous collagen membrane and by adding MTs rather than single cells, we optimize the microenvironment for cell colonization, differentiation and growth. RESULTS & CONCLUSION: Furthermore, in this study, we have shown that by combining BMP-7 with these MSC MTs in this double 3D environment, we further accelerate bone growth in vivo. The strategy described here should enhance the efficiency of therapeutic implants compared with current simplistic approaches used in the clinic today based on collagen implants soaked in bone morphogenic proteins.

Active Nanofibrous Membrane Effects on Gingival Cell Inflammatory Response.

Alpha-melanocyte stimulating hormone (α-MSH) is involved in normal skin wound healing and also has anti-inflammatory properties. The association of α-MSH to polyelectrolyte layers with various supports has been shown to improve these anti-inflammatory properties. This study aimed to evaluate the effects of nanofibrous membrane functionalized with α-MSH linked to polyelectrolyte layers on gingival cell inflammatory response. Human oral epithelial cells (EC) and fibroblasts (FB) were cultured on plastic or electrospun Poly-#-caprolactone (PCL) membranes with α-MSH covalently coupled to Poly-L-glutamic acid (PGA-α-MSH), for 6 to 24 h. Cells were incubated with or without Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). Cell proliferation and migration were determined using AlamarBlue test and scratch assay. Expression of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and transforming growth factor-beta (TGF-ß) was evaluated using RT-qPCR method. Cell cultures on plastic showed that PGA-α-MSH reduced EC and FB migration and decreased IL-6 and TGF-ß expression in Pg-LPS stimulated EC. PGA-α-MSH functionalized PCL membranes reduced proliferation of Pg-LPS stimulated EC and FB. A significant decrease of IL-6, TNF-α, and TGF-ß expression was also observed in Pg-LPS stimulated EC and FB. This study showed that the functionalization of nanofibrous PCL membranes efficiently amplified the anti-inflammatory effect of PGA-α-MSH on gingival cells.

Active Nanomaterials to Meet the Challenge of Dental Pulp Regeneration.

The vitality of the pulp is fundamental to the functional life of the tooth. For this aim, active and living biomaterials are required to avoid the current drastic treatment, which is the removal of all the cellular and molecular content regardless of its regenerative potential. The regeneration of the pulp tissue is the dream of many generations of dental surgeons and will revolutionize clinical practices. Recently, the potential of the regenerative medicine field suggests that it would be possible to achieve such complex regeneration. Indeed, three crucial steps are needed: the control of infection and inflammation and the regeneration of lost pulp tissues. For regenerative medicine, in particular for dental pulp regeneration, the use of nano-structured biomaterials becomes decisive. Nano-designed materials allow the concentration of many different functions in a small volume, the increase in the quality of targeting, as well as the control of cost and delivery of active molecules. Nanomaterials based on extracellular mimetic nanostructure and functionalized with multi-active therapeutics appear essential to reverse infection and inflammation and concomitantly to orchestrate pulp cell colonization and differentiation. This novel generation of nanomaterials seems very promising to meet the challenge of the complex dental pulp regeneration.

Nanostructured thick 3D nanofibrous scaffold can induce bone.

Designing unique nanostructured biomimetic materials is a new challenge in modern regenerative medicine. In order to develop functional substitutes for damaged organs or tissues, several methods have been used to create implants able to regenerate robust and durable bone. Electrospinning produces nonwoven scaffolds based on polymer nanofibers mimicking the fibrillar organization of bone extracellular matrix. Here, we describe a biomimetic 3D thick nanofibrous scaffold obtained by electrospinning of the biodegradable, bioresorbable and FDA-approved polymer, poly(ε-caprolactone). Such scaffold presents a thickness reaching one centimeter. We report here the demonstration that the designed nanostructured implant is able to induce in vivo bone regeneration.