Xavier Garric

Effect of various additives and polymers on lysozyme release from PLGA microspheres prepared by an s/o/w emulsion technique.

Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a two-dimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.

Multilayer, degradable coating as a carrier for the sustained release of antibiotics: preparation and antimicrobial efficacy in vitro.

One of the most critical post-surgical complications is mesh-related infection. This paper describes how a commercially available polypropylene (PP) mesh was modified to minimize the risk of post-implantation infection. A dual drug-release coating was created around mesh filaments using an airbrush spray system. This coating was composed of three layers containing ofloxacin and rifampicin dispersed in a degradable polymer reservoir made up of [poly(ε-caprolactone) (PCL) and poly(DL-lactic acid) (PLA)]. Drug release kinetics were managed by varying the structure of the degradable polymer and the multilayer coating. In vitro, this new drug delivery polymer system was seen to be more rapidly invaded by fibroblasts than was the initial PP mesh. Active mesh showed excellent antibacterial properties with regard to microorganism adhesion, biofilm formation and the periprosthetic inhibition of bacterial growth. Sustained release of the two antibiotics from the coated mesh prevented mesh contamination for at least 72 h. This triple-layer coating technology is potentially of great interest for it can be easily extrapolated to other medical devices and drug combinations for the prevention or treatment of other diseases.

Protein-loaded PLGA–PEG–PLGA microspheres: A tool for cell therapy

A promising strategy to repair injured organs is possible by delivering a growth factor via poly-(d,l lactide-co-glycolide) (PLGA) microspheres; the latter are coated with adhesion molecules that serve as a support for cell delivery. At present, PLGA is not the optimal choice of polymer because of poor or incomplete protein release. The use of a more hydrophilic PLGA-PEG-PLGA (A-B-A) copolymer increases the degree of protein release. In this work, the impact of different combinations of (B) and (A) segments on the protein-release profile has been investigated. Continuous-release profiles, with no lag phases, were observed. The triblock ABA with a low molecular weight of PEG and a high molecular weight of PLGA showed an interesting release pattern with a small burst (<10% in 48 h) followed by sustained, protein release over 36 days. Incomplete protein release was found to be due to various causes: protein adsorption, protein aggregation and protein denaturation under acidic conditions. Interestingly, cell viability and cell adhesion on microspheres coated with fibronectin highlight the interest of these polymers for tissue engineering applications.

New PLGA-P188-PLGA matrix enhances TGF-β3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells.

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest in various clinical applications because it allows cell implantation through minimally invasive surgical procedures. In case of cartilage repair, a tissue engineered construct should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable poly(d,l-lactide-co-glycolide acid) (PLGA), are a unique system, which combines these properties in an adaptable and simple microdevice. However, a limitation of such scaffold is low and incomplete protein release that occurs using the hydrophobic PLGA based microspheres. To circumvent this problem, we developed a novel formulation of polymeric PAMs containing a P188 poloxamer, which protects the protein from denaturation and may positively affect chondrogenesis. This poloxamer was added as a free additive for protein complexation and as a component of the scaffold covalently linked to PLGA. This procedure allows getting a more hydrophilic scaffold but also retaining the protective polymer inside the microcarriers during their degradation. The novel PLGA-P188-PLGA PAMs presenting a fibronectin-covered surface allowed enhanced MSC survival and proliferation. When engineered with TGFβ3, they allowed the sustained release of 70% of the incorporated TGF-β3 over time. Importantly, they exerted superior chondrogenic differentiation potential compared to previous FN-PAM-PLGA-TGF-β3, as shown by an increased expression of specific cartilage markers such as cartilage type II, aggrecan and COMP. Therefore, this microdevice represents an efficient easy-to-handle and injectable tool for cartilage repair.

Calcium-strontium mixed phosphate as novel injectable and radio-opaque hydraulic cement.

Sterile calcium hydrogenophosphate dihydrate (DCPD) (CaHPO(4).2H(2)O), calcium oxide and strontium carbonate powders were mixed in various liquid phases. Among these, ammonium phosphate buffer (0.75 M, pH 6.9) led to a novel strontium-containing calcium phosphate cement. At a 6/2.5/1.5 M ratio and for a liquid to powder ratio (L/P) of 0.5 mlg(-1), the initial paste was fluid and remained injectable for 12 min at 25 degrees C. It was easily obtained by mixing sterile powders and the liquid phase using the push-pull technique, avoiding complex mixing apparatus. The cement set after 15 min at 37 degrees C and was hard after 1h. The compressive strength was in the 20 MPa range, a value higher than that generally assigned to trabecular bone (5-15MPa). This strength appeared sufficient for repairing non-loading sites or reinforcing osteoporotic vertebrae (vertebroplasty). After setting, the initial mixture formed a strontium-calcium-deficient carbonate apatite. The radio-opacity of the resulting cement was three times greater than that of cortical bone because of the presence of strontium ions, a feature that complies with the requirements for vertebroplasty. Furthermore, the cement powder remained stable and retained its properties for at least 4 years.

New antibiotic-eluting mesh used for soft tissue reinforcement.

The surgical implantation of prostheses for soft tissue repair may be followed by post-operative mesh-related infection, a significant and dramatic complication, that is treated by mesh removal. A new antibiotic-eluting mesh has been manufactured on pre-existing polypropylene prostheses using an airbrush spraying technology. Among the degradable polymers tested as coating agents and drug reservoirs, poly(ε-caprolactone) (PCL), which is deposited after heating, provides a homogeneous, regular and smooth shell around the polypropylene filaments of the mesh without dramatically altering the biomechanical properties of the new modified mesh. An anti-infective drug (e.g. ofloxacin) is incorporated into this polymeric coating giving a limited burst effect followed by sustained drug diffusion for several days. An ofloxacin-eluting mesh has demonstrated excellent antibacterial activity in vitro on Escherichia coli adherence, biofilm formation and inhibitory diameter, even with low drug loads. Although further in vivo investigations are required to draw conclusions on the anti-infective effectiveness of the coated mesh, the airbrush coating of ofloxacin-PCL on existing prostheses is already potentially appealing in an effort to decrease post-operative infection.

PLA-based biodegradable and tunable soft elastomers for biomedical applications

Although desirable for biomedical applications, soft degradable elastomers having balanced amphiphilic behaviour are rarely described in the literature. Indeed, mainly highly hydrophobic elastomers or very hydrophilic elastomers with hydrogel behaviours are found. In this work, we developed thermoset degradable elastomers based on the photo-cross-linking of poly(lactide)-poly(ethylene glycol)-poly(lactide) (PLA-PEG-PLA) triblock prepolymers. The originality of the proposed elastomers comes from the careful choice of the prepolymer amphiphilicity and from the possible modulation of their mechanical properties and degradation rates provided by cross-linkers of different nature. This is illustrated with the hydrophobic and rigid 2,4,6-triallyloxy-1,3,5-triazine compared to the hydrophilic and soft pentaerythritol triallyl ether. Thermal properties, mechanical properties, swelling behaviours, degradation rates and cytocompatibility have been evaluated. Results show that it is possible to generate a family of degradable elastomers covering a broad range of properties from a single biocompatible and biodegradable prepolymer.

Is degradable antibiotic coating for synthetic meshes provide protection against experimental animal infection after fascia repair

The surgical repair of pelvic organ prolapse using synthetic mesh can fail because of slow or partial implant integration due to poor biocompatibility or infection. As systemic antibiotic prophylaxis has only limited success, we have developed a system that coats standard polypropylene mesh with clinically relevant antibiotics. Amoxicillin and ofloxacin are both released from the mesh in vitro at high levels over 3 days, preventing adhesion and biofilm formation by a clinical isolate of E. coli. In an in vivo incisional hernia repair model in rats, the antibiotic-coated mesh results in appropriate tissue integration with adequate vascularization and collagen formation. When implanted animals are infected with virulent E. coli, both antibiotic coatings provide full protection against infection (as assessed both clinically and microbiologically), thus demonstrating their bioavailability. This method is a specific approach for producing a therapeutic coating that could reduce postsurgical infections.

Growth of various cell types in the presence of lactic and glycolic acids: the adverse effect of glycolic acid released from PLAGA copolymer on keratinocyte proliferation

Poly(α-hydroxy-acid)s derived from lactic acid (LA) and glycolic acid (GA) are bioresorbable polymers that are currently used in human surgery and in pharmacology to make temporary therapeutic devices. Nowadays, increasing attention is paid to these polymers in the field of tissue engineering. However, the literature shows that a large number of factors can affect many of their properties and the responses of biological systems. As part of our investigation of the biocompatibility of degradable aliphatic polyesters, the effects of LA and GA on the proliferation of various cells under in vitro cell culture conditions were studied. The release of LA and GA from films made of a copolymer synthesized by the zinc lactate method and composed of 37.5% L-lactyl, 37.5% D-lactyl, and 25% glycolyl repeating units was first investigated over a period of 30 days under abiotic conditions in a cell culture medium in order to identify a range of acid concentrations consistent with releases to be expected in real cell cultures. Four cell lines, namely 3T3-J2, C3H101/2, A431, and HaCat, and three primary cell cultures, namely rat endothelial cells, rat smooth muscle cells, and human dermal fibroblasts, were then allowed to grow in the presence of LA and GA at various concentrations taken within the selected 10–1000 mg/cm3 range. Little or no effect was observed on the proliferation of all cells except human keratinocytes, whose growth was dramatically inhibited by GA at concentrations as low as 10 mg/cm3. The inhibiting effect of GA was confirmed by considering the growth of keratinocytes on films made of the same copolymer, in comparison with poly(DL-lactic acid) and polystyrene taken as references. This work shows that GA-releasing degradable matrices are not adapted to the culture of keratinocytes with the aim of making skin grafts.

Potential of a PLA–PEO–PLA-Based Scaffold for Skin Tissue Engineering: In Vitro Evaluation

Abstract This study aimed to investigate the in vitro behaviour of porous degradable scaffolds of the PLA–PEO–PLA-type designed prior to in vivo evaluation for skin tissue engineering. Two tri-block co-polymers were synthesized from PEO and DL-lactide and their degradation was studied under conditions that mimic a cutaneous wound environment. 3-D porous scaffolds with interconnected pores were fabricated using the salt leaching method and characterized by ESEM and Hg porosimetry. The degrading action of gamma sterilization was studied on the co-polymers. The less degraded one was selected to make porous scaffolds on which human dermal fibroblasts and human epidermal keratinocytes were cultured. The capacity of such scaffolds to act as a dermal equivalent was also considered. Colonization by human dermal fibroblasts was shown after hematoxylin staining and the production of major proteins normally found in the extracellular matrix was assessed by Western blotting of protein extracts. Finally, a skin substitute was generated by seeding human keratinocytes on the dermal equivalent and a new epidermis was characterized by using immuno-histological staining. Results show that gamma sterilization and that degradation under conditions that mimic skin wound healing were acceptable. The fact that fibroblasts produce extracellular matrix and that keratinocytes generated an epidermal barrier argues in favour of the interest of this type of porous scaffold for skin reconstruction.