An innovative method to obtain porous PLLA scaffolds with highly spherical and interconnected pores

Scaffolding is an essential issue in tissue engineering and scaffolds should answer certain essential criteria: biocompatibility, high porosity, and important pore interconnectivity to facilitate cell migration and fluid diffusion. In this work, a modified solvent castingparticulate leaching out method is presented to produce scaffolds with spherical and interconnected pores. Sugar particles (200–300 lm and 300–500 lm) were poured through a horizontal Meker burner flame and collected below the flame. While crossing the high temperature zone, the particles melted and adopted a spherical shape. Spherical particles were compressed in plastic mold. Then, poly-L-lactic acid solution was cast in the sugar assembly. After solvent evaporation, the sugar was removed by immersing the structure into distilled water for 3 days. The obtained scaffolds presented highly spherical interconnected pores, with interconnection pathways from 10 to 100 lm. Pore interconnection was obtained without any additional step. Compression tests were carried out to evaluate the scaffold mechanical performances. Moreover, rabbit bone marrow mesenchymal stem cells were found to adhere and to proliferate in vitro in the scaffold over 21 days. This technique produced scaffold with highly spherical and interconnected pores without the use of additional organic solvents to leach out the porogen.

Inplante kirurgiko erradiopakoen bideragarritasuna: plla/bario sulfato sistemaren ``in vitro´´ degradazioa.

[EU]Polimero biobateragarri eta biodegradagarrien erabilera medikuntza arloan aurrerapauso handia suposatu du. Inplanteen arloan (torlojuak, iltzeak, plakak, eta abar…) eta ehun ingeniaritzan garrantzia handia dute. Hala ere, polimero hauen inplanteek desabantaila nabaria aurkezten dute ohiko material metalikoekin alderatuz: erradiopazitate eza. Ikerketa lan honetan karga erradiopako baten adizioak polimero biobateragarri eta biodegradagarri baten giza gorputzaren baldintzetan (pH=7,2 eta 37ºC) burututako in vitro degradazioan duen eragina aztertu da, %70 poli(L-laktida) (PLLA) eta %30 bario sulfato (BaSO4) sistemaren degradazioa, hain zuzen ere. Aztertutako karga erradiopakoak PLLAren degradazioan eragin handirik ez duela ondorioztatu da, beraz, sistema bideragarria dela.

Synthesis, self-assembly in water, and cytotoxicity of MPEG-block-PLLA/DX conjugates

Docetaxel (DX) is one of the most effective antineoplastic drugs. Its current clinical administration is limited because of its hydrophobicity and Serious side effects. A polymer/DX conjugate is designed and successfully prepared to solve these problems. It is monomethoxy-poly(ethylene glycol)-block-poly(L-lactide)/DX (MPEG-PLLA/DX) It was synthesized by reacting DX with carboxyl-terminated copolymer MPEG-PLLA, which was prepared by reacting succinic anhydride with hydroxyl-terminated copolymer monomethoxy-poly(ethylene glycol)-block-poly (L-lactide) (MPEG-PLLA). Its structure and molecular weight was confirmed by H-1 NMR and GPC. The MPEG-PLLA/DX micelles in aqueous solution were prepared Using a SO]vent displacement method and characterized by dynamic light scattering for size and size distribution, and by transmission electron microscopy for surface morphology. Its antitumor activity against HeLa cancer cells evaluated by MTT assay showed that it had a similar antitumor activity to Pure D at the same drug content.

PLLA-PCys co-electrospun fibers for capture and elution of glutathione S-transferase

The copolymer poly(L-lactic acid)-b-poly(L-cysteine) (PLA-b-PCys) was co-electrospun with PLGA into ultrafine fibers. The reduced glutathione (GSH) was conjugated to the fiber surfaces via disulfide bonds. The glutathione S-transferase (GST) was captured onto the GSH fibers via specific substrate-enzyme interaction between the bound GSH and GST. The captured GST was eluted with free GSH aqueous solution and lyophilized to get pure GST powders. The results show that the GSH moieties on the fiber surface retain the bioactivity of the free GSH and thus they can bind specifically with GST and the GST in solution is captured onto the fiber surface. In addition, the bound GSH is not as active as free GSH so that the captured GST can be eluted off from the fiber by free GSH aqueous solution. Based on this principle, GST itself or its fused proteins can be separated and purified very easily. The preliminary purification efficiency is 6.5 mg center dot(g(PCys))(-1). Further improvements are undertaken.

BCNU-loaded PEG-PLLA ultrafine fibers and their in vitro antitumor activity against glioma C6 cells

The purpose of the present study was to develop implantable BCNU-toaded poly(ethylene glycol)poly(L-lactic acid) (PEG-PLLA) diblock copolymer fibers for the controlled release of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was well incorporated and dispersed uniformly in biodegradable PEG-PLLA fibers by using electrospinning method. Environmental Scanning Electron Microscope (ESEM) images indicated that the BCNU-loaded PEG-PLLA fibers looked uniform and their surfaces were reasonably smooth. Their average diameters were below 1500 nm. The release rate of BCNU from the fiber mats increased with the increase of BCNU loading amount. In vitro cytotoxicity assay showed that the PEG-PLLA fibers themselves did not affect the growth of rat Glioma C6 cells. Antitumor activity of the BCNU-loaded fibers against the cells was kept over the whole experiment process, while that of pristine BCNU disappeared within 48 h. These results strongly suggest that the BCNU/PEG-PLLA fibers have an effect of controlled release of BCNU and are suitable for postoperative chemotherapy of cancers.

The potential of electron beam radiation for simultaneous surface modification and bioresorption control of PLLA

Bioresorbable polymers have been widely investigated as materials exhibiting significant potential for successful application in the fields of tissue engineering and drug delivery. Further to the ability to control degradation, surface engineering of polymers has been highlighted as a key method central to their development. Previous work has demonstrated the ability of electron beam (e-beam) technology to control the degradation profiles and bioresorption of a number of commercially relevant bioresorbable polymers (poly-l-lactic acid (PLLA), Llactide/DL-lactide co-polymer (PLDL) and poly(lactic-co-glycolic acid (PLGA)). This work investigates the further potential of ebeam technology to impart added biofunctionality through the manipulation of polymer (PLLA) surface properties. PLLA samples were subjected to e-beam treatments in air, with varying beam energies and doses. Surface characterization was then performed using contact angle analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy. Results demonstrated a significant increase in surface wettability post e-beam treatment. In correlation with this, XPS data showed the introduction of oxygen-containing functional groups to the surface of PLLA. Raman spectroscopy indicated chain scission in the near surface region of PLLA (as predicted). However, e-beam effects on surface properties were not shown to be dependent on beam energy or dose. E-beam irradiation did not seem to affect the surface roughness of PLLA as a direct consequence of the treatment.

The potential of electron beam irradiation for simultaneous surface modification and bioresorption control of PLLA for medical device applications

Bioresorbable polymers have been widely investigated as materials exhibiting significant potential for successful application in the medical fields of bone fixation devices and drug delivery. Further to the ability to control degradation, surface engineering of polymers has been highlighted as a key method central to their development. Previous work has demonstrated the ability of electron beam (e-beam) technology to control the degradation profiles and bioresorption of a number of commercially relevant bioresorbable polymers (poly-l-lactic acid (PLLA), L-lactide/ DL-lactide co-polymer (PLDL) and poly(lactic-co-glycolic acid) (PLGA). This work investigates the further potential of e-beam technology to impart added biofunctionality through the manipulation of polymer (PLLA) surface properties. A Dynamatron Continuous DC e-beam unit (Synergy Health, UK), with beam energies of 0.5, 0.75, and 1.5 MeV, was used for the irradiation of PLLA samples with delivered surface doses of 150 or 500 kGy at each energy level. The chosen conditions reflect the need to achieve a specific surface modification for the control of surface degradation as demonstrated in previous work. Surface characterization was then performed using contact angle analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy.
Results demonstrated a significant increase in surface wettability post e-beam treatment. In correlation with this, XPS data showed the introduction of oxygen-containing functional groups to the surface of PLLA. Raman spectroscopy indicated chain scission in the near surface region of PLLA. E-beam irradiation did not seem to affect the surface roughness of PLLA as a direct consequence of the treatment. In conclusion electron beam surface modification has been found to modify both the surface-to-bulk bioresorption profile and the surface hydrophilicity. Both could provide benefits in relation to the performance of implantable medical devices.

Enhancing the crystallization and orientation of electrospinning poly (lactic acid) (PLLA) by combining with additives

PLLA is a thermoplastic biopolymer and can be used in industrial applications for medical and filtration applications. The brittleness of PLLA is attributed to slow crystallization rates and its glass transition temperature (Tg) is high (60 °C); for this reason, its applications are limited. The orientation, morphology, and crystal structure of the electrospun fibers was investigated by SEM, POM, DSC, FTIR, XRD, and SAXS. Combining with additives leads to a large decrease of fiber diameter, viscosity, and changes of fiber morphology and crystal structure compared to pure PLLA. DSC showed that the Tg of PLLA decreased about 15 °C and there was no change in relaxation enthalpy by the addition of plasticizer. FT-IR indicate a strong interaction between PLLA and additives; a new band appears in the PLLA blend at 1,756 cm−1 at room temperature as a crystalline band without any annealing. In addition, WAXD indicated that the intensities of the two peaks at (200/110) and (203) increased for the blend at room temperature without any annealing in comparison with PLLA; this means that PHB crystallizes in the amorphous region of PLLA. The POM experiments agree with the results from DSC, FTIR, and WAXS measurements, confirming that adding PHB results in an increase in the number of nuclei with much smaller spherulites and enhances the crystallization behavior of this material, thereby improving its potential for applications.

Osteointegration of poly(L-lactic acid)PLLA and poly(L-lactic acid)PLLA/poly(ethylene oxide)PEO implants in rat tibiae

Natural or synthetic materials may be used to aid tissue repair of fracture or pathologies where there has been a loss of bone mass. Polymeric materials have been widely studied, aiming at their use in orthopaedics and aesthetic plastic surgery. Polymeric biodegradable blends formed from two or more kinds of polymers could present faster degradation rate than homopolymers. The purpose of this work was to compare the biological response of two biomaterials: poly(L-lactic acid)PLLA and poly(L-lactic acid)PLLA/poly(ethylene oxide)PEO blend. Forty four-week-old rats were divided into two groups of 20 animals, of which one group received PLLA and the other PLLA/PEO implants. In each of the animals, one of the biomaterials was implanted in the proximal epiphysis of the right tibia. Each group was divided into subgroups of 5 animals, and sacrificed 2, 4, 8 and 16 weeks after surgery, respectively. Samples were then processed for analysis by light microscopy. Newly formed bone was found around both PLLA and PLLA/PEO implants. PLLA/PEO blends had a porous morphology after immersion in a buffer solution and in vivo implantation. The proportion 50/50 PLLA/PEO blend was adequate to promote this porous morphology, which resulted in gradual bone tissue growth into the implant.

Crystallization and morphology of the PLLA phase within random poly (L-lactide-ran-ɛ-caprolactone)

This work has mainly focused on the poly (L-lactide) (PLLA) which is a material for multiple applications with performances comparable to those of petrochemical polymers (PP, PS, PET, etc. ...), readily recyclable and also compostable. However, PLLA has certain shortcomings that limit its applications. It is a brittle, hard polymer with a very low elongation at break, hydrophobic, exhibits low crystallization kinetics and takes a long time to degrade. The properties of PLLA may be modified by copolymerization (random, block, and graft) of L-lactide monomers with other co-monomers. In this thesis it has been studied the crystallization and morphology of random copolymers poly (L-lactide-ran-ε-caprolactone) with different compositions of the two monomers since the physical, mechanical, optical and chemical properties of a material depend on this behavior. Thermal analyses were performed by differential scanning calorimetry (DSC) and thermogravimetry (TGA) to observe behaviors due to the different compositions of the copolymers. The crystallization kinetics and morphology of poly (L-lactide-ran-ε-caprolactone) was investigated by polarized light optical microscopy (PLOM) and differential scanning calorimetry (DSC). Their thermal behavior was observed with crystallization from melt. It was observed that with increasing amounts of PCL in the copolymer, there is a decrease of the thermal degradation. Studies on the crystallization kinetics have shown that small quantities of PCL in the copolymer increase the overall crystallization kinetics and the crystal growth rate which decreases with higher quantities of PCL.

Synthesis, characterization and crystallization of bionanocomposites based on PLLA, PCL and nanocellulose

In recent years, environmental concerns and the expected shortage in the fossil reserves have increased further development of biomaterials. Among them, poly(lactide) PLA possess some potential properties such as good ability process, excellent tensile strength and stiffness equivalent to some commercial petroleum-based polymers (PP, PS, PET, etc.). This biobased polymer is also biodegradable and biocompatible However, one great disadvantage of commercial PLA is slow crystallization rate, which restricts its use in many fields. Using of nanofillers is viewed as an efficient strategy to overcome this problem. In this thesis, the effect of bionanofillers in neat PLA and in blends of poly (L-lactide)(PLA)/poly(ε-Caprolactone) (PCL) has been investigated. The used nanofillers are: poly(L-lactide-co-ε-caprolactone) and poly(L-lactide-b-ε-caprolactone) grafted on cellulose nanowhiskers and neat cellulose nanowhiskers (CNW). The grafting reaction of poly(L-lactide-co-caprolactone) and poly (L-lactide-b-caprolactone) on the nanocellulose has been performed by the grafting from technique. In this way the polymerization reaction it is directly initiated on the substrate surface. The condition of the reaction were chosen after a temperature and solvent screening. By non-isothermal an isothermal DSC analysis the effect of bionanofillers on PLA and 80/20 PLA/PCL was evaluated. Non-isothermal DSC scans show a nucleating effect of the bionanofillers on PLA. This effect is detectable during PLA crystallization from the glassy state. Cold crystallization temperature is reduced upon the addition of the poly(L-lactide-b-caprolactone) grafted on cellulose nanowhiskers that is most performing bionanofiller in acting as a nucleating agent. On the other hand, DSC isothermal analysis on the overall crystallization rate indicate that cellulose nanowhiskers are best nucleating agents during isothermal crystallization from the melt state. In conclusion, nanofillers have different behavior depending on the processing conditions. However, the efficiency of our nanofillers as nucleating agent was clearly demonstrated in both isothermal as in non-isothermal condition.

Bionanocomposites based on Plla, Pcl and montmorillonite: synthesis, characterization and crystallization

Poly(lactide) is one of the best candidate to replace conventional petroleum-based polymers, since it is biobased, biocompatible and biodegradable. However, commercial PLA materials typically have low crystallization rate resulting in long processing time and low production efficiency. In this work the effects of two nanofillers MMT30B and MMT30B-g-P(LA-co-CL) on the crystallization rate of neat PLA and PLA/PCL blend were investigated. MMT30B-g-P(LA-co-CL) was synthetized by in situ grafting reaction. The synthesis was carried in xylene at 140°C, upon the results of a screening. The grafted copolymers were evaluated by 1H-NMR ,ATR–IR and TGA. Solvent casted films were obtained by mixing MMT30B-g-P(LA-co-CL) at 5% (w/w) with neat PLA and PLA/PCL blend, comparing the properties with the corresponding blends with and without a 5% of (w/w) unmodified clay. SEM images on PLA based blends shows that MMT30B is aggregated into larger particles compared to MMT30B-g-P(LLA-co-CL). This behavior is correlated to the better exfoliation of MMT30B-g-P(LA-co-CL) clay layers. SEM images on PLA/PCL based blends exhibit the typical sea-island morphology, characteristic of immiscible blends. PLA is the matrix while PCL is finely dispersed in droplets. MMT30B does not reduce PCL droplets size, while MMT30B-g-P(LA-co-CL) reduces the size of PCL droplets. This means that MMT30B-g-P(LA-co-CL) can migrate to the PLA-PCL interface, acting as a compatibilizer. Non-isothermal DSC cooling scans show a fractionated crystallization of the PCL phase in PLA/PCL/MMT30B-g-P(LA-co-CL), confirming the compatibilizer effect of MMT30B-g-P(LA-co-CL). At the same timeMMT30B-g-P(LA-co-CL) can better nucleate the PLA phase, both in neat PLA and PLA/PCL blend, promoting the crystallization during the heating scans. In isothermal condition, both the nanofillers increase the crystallization rate of PLA phase in neat PLA, while in PLA/PCL blends the effect is covered by the nucleating effect of PCL.

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.