Esther Sánchez

In vitro-in vivo characterization of gentamicin bone implants.

A gentamicin carrier system composed of calcium phosphates, poly(DL-lactide) (PLA) and gentamicin was developed and characterized in vitro and in vivo for use in the prevention and treatment of bone infection. Four formulations were prepared according to an experimental design based on the Hadamard matrix. The technological variables included in the design were: gentamicin loading with respect to the implant weight, weight average molecular weight (M(w)) of the PLA as a compound of the matrix and the presence or absence of a PLA coating of 200 kDa. The variable to be optimized in vitro was the gentamicin release level during the first week. According to this goal, the selected formulation was F-D which was composed of 80% phosphates (25% hydroxyapatite, HAP and 75% tricalcium phosphate, TCP), 20% PLA (M(w), 30 kDa) and 3.5% gentamicin sulfate (GS) and was coated with PLA (M(w), 200 kDa). To elucidate the in vitro release mechanism of this implant, another implant lot (F-X) uncoated, but with identical matrix composition, was prepared. Results showed that the PLA coating delay the gentamicin release, indicating that part of the antibiotic released from the matrix diffuses through the polymer coating film. The selected formulation was tested in the femur of rabbits and showed a faster release rate in vivo than in vitro. This is due to a greater degree of PLA degradation, changes in the phosphate blend, and bone tissue invading the implant. Gentamicin concentration in the areas of the bone closest to the implant was higher than the minimum inhibitory concentration (MIC) against Staphylococcus aureus.

Local controlled release of VEGF and PDGF from a combined brushite-chitosan system enhances bone regeneration.

The two growth factors VEGF and PDGF are involved in the process of bone regeneration. For this reason, we developed a brushite-chitosan system which controls the release kinetics of incorporated VEGF and PDGF to enhance bone healing. PDGF (250 ng) was incorporated in the liquid phase. Alginate microsphere-encapsulated VEGF (350 ng) was pre-included in small cylindrical chitosan sponges. VEGF and PDGF release kinetics and tissue distribution were determined using iodinated ((125)I) growth factor. In vivo, PDGF was more rapidly delivered from these systems implanted in rabbit femurs than VEGF. 80% of PDGF was released by the end of two weeks while only 70% of VEGF was delivered after a period of three weeks. Both GFs released from the brushite-chitosan constructs remained located around the implantation site (5 cm) with negligible systemic exposure. A PDGF bone peak concentration of approximately 5 ng/g was achieved on the 4th day. Thereafter, PDGF concentrations stayed higher than 2 ng/g during the first week. These scaffolds also provided a local VEGF bone concentration above 3 ng/g during a total of 4weeks, with a peak concentration of 5.5 ng/g on the 7th day. The present work demonstrates that our brushite-chitosan system is capable of controlling the release rate and localization of both GFs within a bone defect. The effect on bone formation was considerably enhanced with PDGF loaded brushite-chitosan scaffolds as well as with the PDGF/VEGF combination.

VEGF-controlled release within a bone defect from alginate/chitosan/PLA-H scaffolds.

VEGF and its receptors constitute the key signaling system for angiogenic activity in tissue formation, but a direct implication of the growth factor in the recruitment, survival and activity of bone forming cells has also emerged. For this reason, we developed a composite (alginate/chitosan/PLA-H) system that controls the release kinetics of incorporated VEGF to enhance neovascularization in bone healing. VEGF release kinetics and tissue distribution were determined using iodinated ((125)I) growth factor. VEGF was firstly encapsulated in alginate microspheres. To reduce the high in vitro burst release, the microspheres were included in scaffolds. Matrices were prepared with alginate (A-1, A-2), chitosan (CH-1, CH-2) or by coating the CH-1 matrix with a PLA-H (30 kDa) film (CH-1-PLA), the latter one optimally reducing the in vitro and in vivo burst effect. The VEGF in vitro release profile from CH-1-PLA was characterized by a 13% release within the first 24h followed by a constant release rate throughout 5 weeks. For VEGF released from composite scaffolds in vitro, bioactivity was maintained above 90% of the expected value. Despite the fact that the in vivo release rate was slightly faster, a good in vitro-in vivo correlation was found. The VEGF released from CH-1 and CH-1-PLA matrices implanted into the femurs of rats remained located around the implantation site with a negligible systemic exposure. These scaffolds provided a bone local GF concentration above 10 ng/g during 2 and 5 weeks, respectively, in accordance to the in vivo release kinetics. Our data show that the incorporation of VEGF into the present scaffolds allows for a controlled release rate and localization of the GF within the bone defect.

In vivo-in vitro study of biodegradable and osteointegrable gentamicin bone implants

Three implants composed of phosphate (25% hydroxyapatite, 75% tricalcium phosphate), 20% poly(DL-lactide) (DL-PLA; weight-average molecular weight (Mw), 30 kD) and 3% gentamicin sulphate (GS) were assayed in vitro and in vivo to study their release profiles as potential drug delivery systems to prevent or treat osteomyelitis. To prolong GS release, some implants were coated with poly(lactide-co-glycolide) (PLGA; Mw, 100 kD; I-PLGA) or DL-PLA (Mw, 200 kD; I-PLA). GS levels were measured in bone, kidney and blood after implantation into the femur of rats. The release profiles show a burst in the first few days, followed by a slower release rate. After I-PLA implantation, bone antibiotic concentrations higher than the minimum bactericidal concentration were maintained for 4 weeks. A linear correlation between in vitro and in vivo GS release was found to continue until complete drug release. Histological and radiological analysis showed that the implants were well tolerated and gradual new bone formation was observed.

Material-related effects of BMP-2 delivery systems on bone regeneration

Material-related effects of a brushite and a PLGA controlled release system loaded with two distinct doses of bone morphogenetic protein-2 (BMP-2) (3.5 and 17.5 μg), pre-encapsulated in poly(lactic-co-glycolic acid) (PLGA), were investigated in an intramedullary femur defect model in rabbits. The systems were characterized in vitro and in vivo over 12 weeks in terms of morphology, release kinetics, porosity, molecular weight, and composition using scanning electron microscopy, mercury porosimetry, radioactivity counting, X-ray diffractometry, differential scanning calorimetry, and gel permeation chromatography. During the experimental period the investigated systems underwent significant changes in vitro as well as in vivo. It should be stressed that the two in vitro release patterns were similar, however in vivo parallel profiles were observed with a higher burst effect for BMP-2 in the PLGA system. The PLGA system degraded and disintegrated significantly faster than the brushite system, which suffered slowly progressing external erosion and, additionally, material resorption by osteoclasts in vivo. The consequences of this were reflected in the degree of bone regeneration. Although a sustained delivery of BMP-2 was achieved with both systems, the brushite construct, independent of the loaded growth factor dose, failed to consistently induce defect repair, a result attributed to its slow resorption rate. In contrast, the PLGA system resulted in complete regeneration with mature trabecular bone formation 8 weeks after implantation.

Radiolabelled biodegradable microspheres for lung imaging.

The effect on lung accumulation of modifying the surface compositions of (99m)Tc poly(lactide-co-glycolide) (PLGA) and (99m)Tc poly(ethylene glycol)-poly(lactide-co-glycolide) (PEG-PLGA) microspheres with different surfactants was assessed after intravenous injection into rats. Microspheres were prepared with PLGA or PEG-PLGA by the emulsion solvent evaporation method using polyvinyl alcohol (PVA), polyethylene glycol (PEG), albumin (BSA) or poloxamer 188 as surfactant, in the external aqueous phase. Commercial human albumin microspheres (Sferotec((R)), HAM) were used as reference. According to the European Pharmacopeia, >80% of (99m)Tc-HAM in the size range 10-50 microm, must be accumulated in the lung 15 min after intravenous administration. By modifying the surfactant, the resulting lung accumulation was 99% for (99m)Tc-HAM, and more than 50% for PLGA microspheres prepared with poloxamer 188 (1 and 4%), reaching 67% with 8% Poloxamer 188 and around 30-39% for PLGA and PEG-PLGA microspheres prepared with the other surfactants. PLGA microspheres made with 8% poloxamer 188 gave good quality lung images under a gamma camera for the first few minutes, subsequently liver radioactivity masked lung images.

Repair of an osteochondral defect by sustained delivery of BMP-2 or TGFβ1 from a bilayered alginate-PLGA scaffold.

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (125I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. Copyright

In vivo osteogenic response to different ratios of BMP-2 and VEGF released from a biodegradable porous system.

Bone regeneration and vascularization with porous PLGA scaffolds loaded with VEGF (0.35 and 1.75 μg) and BMP-2 (3.5 and 17.5 μg), incorporated in PLGA microspheres, or the combination of either dose of BMP-2 with the low dose of VEGF were investigated in an intramedullary femur defect in rabbits. The system was designed to control growth factor (GF) release and maintain the GFs localized within the defect. An incomplete release was observed in vitro whereas in vivo VEGF and BMP-2 were totally delivered during 3 and 4 weeks, respectively. A weak synergistic effect of the dual delivery of VEGF and BMP-2 (high dose) was found by 4 weeks. However, the absence of an apparent synergistic long-term effect (12 weeks) of the combination over BMP-2 alone suggests that more work has to be done to optimize VEGF dose, sequential presentation, and the ratio of the two GFs to obtain a beneficial bone repair response.

Cartilage repair by local delivery of transforming growth factor‐β1 or bone morphogenetic protein‐2 from a novel, segmented polyurethane/polylactic‐co‐glycolic bilayered scaffold

This study aimed to analyze the in vitro and in vivo release kinetics and evaluate the grades of repair induced by either the release of 50 ng of transforming growth factor-β1 or 2.5 or 5 μg of bone morphogenetic protein-2 (BMP-2) from a bilayer scaffold of segmented polyurethane/polylactic-co-glycolic (SPU/PLGA) in osteochondral defects, in a rabbit model. The scaffold consisted of a porous, bone-directed PLGA layer, overlaid with a cartilage-directed layer of growth factor (GF)-loaded PLGA microspheres, dispersed in a matrix of SPU. The PLGA porous layer was fabricated by gas foaming. Microspheres were prepared by a double emulsion method. SPU was synthesized by following the two-step method. GF release kinetics were assessed using iodinated ((125)I) GFs. The in vivo release profiles of both GFs fitted to zero-order kinetics, demonstrating a consistently good control of their release rates by SPU. Cartilage-like tissue, characterized by histological analysis, scoring, and immunolabeling of chondrogenic differentiation markers, was observed only after 12 weeks, maintaining integrity up to at least 24 weeks, independently of the GF and the dose of BMP-2. The biocompatibility and the resulting good quality, hyaline repair cartilage convert this system into a promising candidate for future applications in osteochondral lesions.

Effect of triple growth factor controlled delivery by a brushite-PLGA system on a bone defect.

Bone regeneration is a complex process that involves multiple cell types, growth factors (GFs) and cytokines. A synergistic contribution of various GFs and a crosstalk between their signalling pathways was suggested as determinative for the overall osteogenic outcome. The purpose of this work was to develop a brushite-PLGA system, which controls the release rate of the integrated growth factors (GFs) to enhance bone formation. The brushite cement implants were prepared by mixing a phosphate solid phase with an acid liquid phase. PDGF (250 ng) and TGF-β1 (100 ng) were incorporated into the liquid phase. PLGA microsphere-encapsulated VEGF (350 ng) was pre-blended with the solid phase. VEGF, PDGF and TGF-β1 release kinetics and tissue distributions were determined using iodinated ((125)I) GFs. In vivo results showed that PDGF and TGF-β1 were delivered more rapidly from these systems implanted in an intramedullary defect in rabbit femurs than VEGF. The three GFs released from the brushite-PLGA system remained located around the implantation site (5 cm) with negligible systemic exposure. Bone peak concentrations of approximately 4 ng/g and 1.5 ng/g of PDGF and TGF-β1, respectively were achieved on day 3. Thereafter, PDGF and TGF-β1 concentrations stayed above 1 ng/g during the first week. The scaffolds also provided a VEGF peak concentration of nearly 6 ng/g on day 7 and a local concentration of approximately 1.5 ng/g during at least 4 weeks. Four weeks post implantation bone formation was considerably enhanced with the brushite-PLGA system loaded with each of the three GFs separately as well as with the combination of PDGF and VEGF. The addition of TGF-β1 did not further improve the outcome. In conclusion, the herein presented brushite-PLGA system effectively controlled the release kinetics and localisation of the three GFs within the defect site resulting in markedly enhanced bone regeneration.