Synthesis and characterization of poly(L-lactic acid) membranes: Studies in vivo and in vitro

The use of biodegradable polyesters as temporary structural supports in the recuperation of damaged live tissue is a promising area of research. Poly(L-lactic acid) (PLLA) membranes can act as a support for cell fixation and growth or as a barrier against soft tissues invasion in recuperating bone tissues. In this work, five different types of PLLA membranes, which varied in their polymer–solvent ratio and their content of plasticizer were studied. For the study in vivo, 6 mm diameter disks were inserted subcutaneously in the dorsal region of 15 Wistar rats, and the reactions on rats were studied 15 days later. In another series of experiments the samples were immersed in phosphate buffer, pH 7.4 at 37 °C, for 30 days. Membranes without plasticizer were morphologically dense and did not allow cell invasion nor tissue adherence, in contrast to membranes with plasticizer. While porosity enhanced cell fixation and growth, it made the membrane more fragile mechanically when compared to membranes without pores.     

Immobilization of poly (ethylene imine) on poly (l-lactide) promotes MG63 cell proliferation and function

Poly (ethylene imine) (PEI) is a polycation widely used for DNA transfection to cells but also applied as primary polycation for layer-by-layer (LBL) assembly of polyelectrolytes. The aim of the present study was to investigate the effect of modification with PEI on the biocompatibility of poly (l-lactide) (PLLA) films. PEI with different molecular weight was immobilized on PLLA by either adsorption or covalent binding. Cell morphologies, immuno-fluorescence staining, cell proliferation by lactate dehydrogenase assay and cell differentiation by alkaline phosphatase assay were utilized to assess the biocompatibility of the modified PLLA using osteoblast cell line MG63. Results revealed that PEI modification remarkably improved cell adhesion, viability, proliferation and function compared with plain PLLA. Hence, PEI-modified PLLA is acceptable as transfection vehicle for engineering of bone and other tissues, or as primary layer to allow LBL assembly to generate biomimetic surface coatings.     

Antibacterial and anti-adhesion effects of the silver nanoparticles-loaded poly(l-lactide) fibrous membrane

The complications of tendon injury are frequently compromised by peritendinous adhesions and tendon sheath infection. Physical barriers for anti-adhesion may increase the incidence of postoperative infection. This study was designed to evaluate the potential of silver nanoparticles (AgNPs)-loaded poly(l-lactide) (PLLA) electrospun fibrous membranes to prevent adhesion formation and infection. Results of an in vitro drug release study showed that a burst release was followed by sustained release from electrospun fibrous membranes with a high initial silver content. Fewer fibroblasts adhered to and proliferated on the AgNP-loaded PLLA electrospun fibrous membranes compared with pure PLLA electrospun fibrous membrane. In the antibacterial test, the AgNP-loaded PLLA electrospun fibrous membranes can prevent the adhesion of Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and Gram-negative Pseudomonas aeruginosa. Taken together, these results demonstrate that AgNP-loaded PLLA electrospun fibrous membranes have the convenient practical medical potential of reduction of infection and adhesion formation after tendon injury.     

Needle-like nano hydroxyapatite/poly(l-lactide acid) composite scaffold for bone tissue engineering application

In this paper, a new nano-hydroxyapatite / poly (l-lactide acid) (nHAP/PLLA) composite scaffold comprising needle-like nHAP particles was prepared. In the first step, the identification and morphology of chemically synthesized HAP particles were determined by XRD, EDX, FTIR and SEM analyses. The needle-like nHAP particles with an average size of approximately 30–60 nm in width and 100–400 nm in length were found similar to needle-like bone nano apatites in terms of chemical composition and morphology. In the second step, nHAP and micro-sized HAP (mHAP) particles were used to fabricate HAP filled PLLA (HAP/PLLA) composites scaffolds using solid–liquid phase separation method. The porosity of scaffolds was up to 85%, and their average macropore diameter was in the range of 64–175 µm. FTIR and XRD analyses showed the presence of molecular interactions and chemical linkages between HAP particles and PLLA matrix. The compressive strength of nanocomposite scaffolds could high up to 8.46 MPa while those of pure PLLA and microcomposite scaffolds were 1.79 and 4.61 MPa, respectively. The cell affinity and cytocompatibility of the nanocomposite scaffold were found to be higher than those of pure PLLA and microcomposite scaffolds. Based on the results, the newly developed nHAP/PLLA composite scaffold is comparable with cancellous bone in terms of microstructure and mechanical strength, so it may be a suitable alternative for bone tissue engineering applications.     

Fabrication of poly(lactic acid)-poly(ethylene oxide) electrospun membranes with controlled micro to nanofiber sizes

Biodegradable poly(L-lactide acid) (PLLA) nanofiber membranes were prepared by electrospinning of PLLA and poly(ethylene oxide) (PEO). The selective removal of PEO by water allows to obtain smaller fiber diameters and to increase the porosity of the membranes in comparison to PLLA membranes obtained under the same electrospinning conditions. After removal of PEO membranes with fiber sizes of 260 nm and average porosity close to 80% are obtained. Thermal and infrared results confirm the poor miscibility of PLLA and PEO, with the PEO randomly distributed along the PLLA fibers. On the other, PLLA and PEO mixing strongly affect their respective degradation temperatures. The influence of the PEO in the electrospinning process is discussed and the results are correlated to the evolution of the PLLA fiber diameter.     

Surface modification of poly(l-lactide) electrospun fibers with nanocrystal hydroxyapatite for engineered scaffold applications

The hydrophobicity of the poly(l-lactide) (PLLA) surface was modified by incorporating hydroxyapatite (HAp) nanocrystalline particles during the electrospinning process for the engineered scaffold applications. The HAp nanocrystals were synthesized with 30 nm in diameter and 100–120 nm in length, which subsequently formed micrometer-sized agglomerates in the range of 2.5 μm. The synthesized HAp agglomerates were electrospun in the PLLA solution, and the HAp nanocrystals were desirably exposed on the surface of the electrospun PLLA fibers to give higher surface energy and lower contact angles with water. The surface-exposed hydrophilic HAp nanocrystals substantially increased the precipitation of various salts on the HAp/PLLA fiber surfaces in a buffer solution due to the hydrophilic nature and ionic affinity of HAp. Finally, the developed HAp/PLLA fibers desirably sustained the fibrous structural integrity during the accelerated-aging test in water, which was not the case with the pristine PLLA fibers.     

纳米纤维素晶须增强增韧聚(L-乳酸)复合材料的制备与表征

通过酸解法制备了具有纳米尺寸和一定长径比针棒状的纳米纤维素晶须(NCW),利用NCW表面的羟基引发L-丙交酯开环聚合,合成了表面接枝聚(L-乳酸)(PLLA)链段的接枝纤维素晶须(g-NCW);采用溶液浇铸法制备了PLLA膜以及不同配比的NCW/PLLA和g-NCW/PLLA复合膜。对接枝改性前后的NCW的形貌与性能进行了表征,研究了复合膜的形貌、结晶性能、热稳定性、亲/疏水性和拉伸性能。结果表明:NCW的形貌与结晶性能在接枝改性后变化不大,但在乙醇和PLLA溶液中的分散性得到明显改善;当NCW与L-丙交酯的物质的量之比为1∶5时,g-NCW表面PLLA链段的接枝率约为23.61%。NCW和g-NCW作为异相成核剂,显著提高了PLLA基体的结晶速率;并且,加入晶须改善了材料的亲水性和热稳定性。添加一定量的NCW和g-NCW到PLLA中,可有效增强增韧PLLA基体;随着晶须含量增加,复合膜的拉伸强度和断裂能先增大后下降;当NCW和g-NCW的质量分数为5%时,NCW/PLLA和g-NCW/PLLA复合膜的拉伸强度和断裂能分别达到22.02 MPa和20.01 MPa以及102.39J/m~3和117.83J/m~3,均达到最大值。由于g-NCW在基体中良好的分散性以及与基体间的界面结合,g-NCW/PLLA复合膜的拉伸强度和韧性明显优于相应的纯PLLA和NCW/PLLA膜。     

Effect of lecithin content blend with poly (L-lactic acid) on viability and proliferation of mesenchymal stem cells

Lecithin constitutes a natural mixture of phospholipids and neutral lipids and plays critical roles in cellular membrane structure and cellular signaling. In this study, lecithin was blended with poly (L-lactic acid) (PLLA) for modifying the surface of PLLA because it might obtain appropriate hydrophilicity and biocompatibility. The modified PLLA films were manufactured using conventional solvent-casting technique. The hydrophilicity clearly increased with an increase of lecithin content in the polymer blends, as determined by measuring the water contact angle (WCA). The cytocompatibility and any potential cytotoxic effects were studied over 7 days by seeding mesenchymal stem cells (MSCs) on the films of PLLA containing 0–15% lecithin (wt.%), in comparison with tissue culture plates (TCPs). Cell viability and proliferation were assessed using WST-8, lactate dehydrogenase (LDH) and cell morphology was studied by toluidine blue and propidium iodide staining. This results obtained above suggested that 5%lecithin-containing PLLA films could possess the optimal hydrophilicity, higher adhesion and proliferation of MSCs for a prolonged period and did not demonstrate any significant toxic effects to cells. The study showed that the hydrophilicity and biocompatibility of the modified PLLA were markedly improved by directly introducing lecithin into the polymer without the use of multiple synthetic steps. The information obtained should be useful for future research in vascular tissue engineering (VTE).     

Heat and compression molded electrospun poly(l-lactide) membranes: Preparation and characterization

The fibrous membranes prepared by electrospinning have great advantages, such as high porosity and high specific surface area. However, low mechanical strength of electrospun membranes has been one of the most difficult technical problems to overcome, resulting in negative impact on the application. In this paper, the heat-assisted compression approach was employed to improve the mechanical performances of electrospun poly(l-lactide)(PLLA) membranes, especially in tensile strength. It is found that the electrospun PLLA membranes crystallize in α form and strong fiber-to-fiber linkages occurred with the aid of heat and compression. The tensile properties including tensile strength and modulus of membranes treated with a press at 6 MPa and a temperature at 60 °C (80 °C and 100 °C) increased by more than 100% compared with those of the as-electrospun membranes.     

Three-dimensional traction forces of Schwann cells on compliant substrates

The mechanical interaction between Schwann cells (SCs) and their microenvironment is crucial for the development, maintenance and repair of the peripheral nervous system. In this paper, we present a detailed investigation on the mechanosensitivity of SCs across a physiologically relevant substrate stiffness range. Contrary to many other cell types, we find that the SC spreading area and cytoskeletal actin architecture were relatively insensitive to substrate stiffness with pronounced stress fibre formation across all moduli tested (0.24–4.80 kPa). Consistent with the presence of stress fibres, we found that SCs generated large surface tractions on stiff substrates and large, finite material deformations on soft substrates. When quantifying the three-dimensional characteristics of the SC traction profiles, we observed a significant contribution from the out-of-plane traction component, locally giving rise to rotational moments similar to those observed in mesenchymal embryonic fibroblasts. Taken together, these measurements provide the first set of quantitative biophysical metrics of how SCs interact with their physical microenvironment, which are anticipated to aid in the development of tissue engineering scaffolds designed to promote functional integration of SCs into post-injury in vivo environments.     

In vivo interaction of cells on poly L-(lactic acid) membranes containing plasticizer

The development of biodegradable materials has lead to renewed interest in the study of their interactions with the host organism in order to make the resulting products appropriate for use as temporary materials in protheses. Poly L-(lactic acid)(PLLA)-based biodegradable devices have been used for several purposes. The physical properties of these materials can be modified by the addition of a plasticizer, such as the triethylcitrate, to provide flexibility and porosity to the implants and enhance control of the polymer degradation time. In this work we examined the biological properties of a PLLA porous membrane containing 7% triethylcitrate, by assessing the process of degradation and the interaction with dermal tissue. Samples of skin obtained from female Wistar rats 2–180 days after implantation with PLLA-based membrane were processed for light microscopy and scanning electron microscopy. The membranes became surrounded by a delicate network of connective tissue which gradually invaded the membrane structure. Polymer degradation began with the appearance of radial fractures in the globular units of the biodegradable membrane, especially by 90 and 180 days after implantation.     

Biomimetic poly(glycerol sebacate) (PGS) membranes for cardiac patch application

In this study biomimetic poly(glycerol sebacate) PGS matrix was developed for cardiac patch application. The rationale was that such matrices would provide conducive environment for the seeded cells at the interphase with PGS. From the microstructural standpoint, PGS was fabricated into dense films and porous PGS scaffolds. From the biological aspect, biomimetic PGS membranes were developed via covalently binding peptides Tyr-Ile-Gly-Ser-Arg (YIGSR) and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), corresponding to the epitope sequences of laminin and fibronectin, respectively onto the surface. To improve and enhance homogenous binding of peptides onto the PGS surface, chemical modification of its surface was carried out. A sequential regime of alkaline hydrolysis with 0.01 M NaOH for 5 min and acidification with 0.01 M HCl for 25 s was optimal. More COOH chemical group was exposed without causing deleterious effect on the bulk properties of the polymer as revealed by the physicochemical analysis carried out. HPLC analysis, chemical imaging and ToF-SIMS were able to establish the successful homogenous functionalization of PGS membranes with the peptides. Finally, the developed biomimetic membranes supported the adhesion and growth of rat and human cardiac progenitor cells.     

聚乳酸复合纳米纤维创面敷料的制备及性能

采用静电纺丝技术制备了聚乳酸(PLLA)纳米纤维毡、壳聚糖/PLLA纳米纤维毡和明胶/PLLA纳米纤维毡。利用扫描电镜(SEM)、图像分析软件等手段研究了纳米纤维微观形貌,并研究各种创面敷料的吸水性、保水性和水蒸汽通透性等性能。结果表明,壳聚糖/PLLA、明胶/PLLA复合纳米纤维毡的吸水性和保水性有显著提高,水蒸汽通透性略有下降,是理想的创面敷料材料。     

Biodegradation of porous versus non-porous poly(L-lactic acid) films

The influence of porosity on the degradation rate of poly(L-lactic acid) (PLLA) films was investigated in vitro and in vivo. Non-porous, porous and combi (porous with a non-porous layer) PLLA films were used. Changes in Mw, Mn, polydispersity (Mw/Mn) ratio, melting temperature (T _m), heat of fusion, tensile strength, E-modulus, mass and the remaining surface area of cross-sections of the PLLA films were measured. In general, during the degradation process, the porous film has the highest Mw, Mn, Mw/Mn ratio and T _m, while the non-porous film has the lowest. In contrast, the highest heat of fusion values were observed for the non-porous film, indicating the presence of relatively smaller molecules forming crystalline domains more easily. The tensile strength and E-modulus of the non-porous film decrease faster than those of the porous and the combi film. None of the three types of films showed massive mass loss in vitro nor a significant decrease in remaining polymer surface area in light microscopical sections in vitro and in vivo. Heavy surface erosion of the non-porous layer of the combi film was observed after 180 days, turning the combi film into a porous film. This is also indicated by the changes in tensile strength, Mw, Mw/Mn, T _m and heat of fusion as a function of time. It is concluded that non-porous PLLA degrades faster than porous PLLA. Thus, in our model, porosity is an important determinant of the degradation rate of PLLA films.     

Formation of bone-like apatite on poly(L-lactide) to improve osteoblast-like compatibility in vitro and in vivo

The biomimetic apatite coating process was adopted to modify poly(L-lactide) (PLLA) surfaces with osteoblasts-like cell compatibility. The apatite coating was formed on the pre-hydrolyzed PLLA film and scaffold surfaces by incubating in simulated body fluid (SBF). Scanning electron microscopy and energy dispersive X-ray analyzer were utilized to characterize the composition and the structure of the apatite coating. The cytocompatibility of the modified PLLA films was investigated by testing osteoblast-like attachment, proliferation, alkaline phosphatase (ALP) activity, and cell cycle. Subsequently, the modified PLLA scaffolds were co-cultured with the osteoblasts-like in vitro and subcutaneously implanted into nude mice. The experimental results showed that the formed apatite had a nano-sized particle and matrix configuration. The surface modification of PLLA with apatite coating significantly promoted osteoblast-like compatibility. After a four-week culture in vivo, no significant inflammatory signs were observed in the implanted regions and osteoblast-like congeries with bone-like structure began to form in the scaffolds. The positive results of this study suggest a good way to produce desirable PLLA biomaterials for bone tissue engineering.     

Electrospun magnetic poly(l-lactide) (PLLA) nanofibers by incorporating PLLA-stabilized Fe3O4 nanoparticles

Magnetic poly(l-lactide) (PLLA)/Fe₃O₄ composite nanofibers were prepared with the purpose to develop a substrate for bone regeneration. To increase the dispersibility of Fe₃O₄ nanoparticles (NPs) in the PLLA matrix, a modified chemical co-precipitation method was applied to synthesize Fe₃O₄ NPs in the presence of PLLA. Trifluoroethanol (TFE) was used as the co-solvent for all the reagents, including Fe(II) and Fe(III) salts, sodium hydroxide, and PLLA. The co-precipitated Fe₃O₄ NPs were surface-coated with PLLA and demonstrated good dispersibility in a PLLA/TFE solution. The composite nanofiber electrospun from the solution displayed a homogeneous distribution of Fe₃O₄ NPs along the fibers using various contents of Fe₃O₄ NPs. X-ray diffractometer (XRD) and vibration sample magnetization (VSM) analysis confirmed that the co-precipitation process had minor adverse effects on the crystal structure and saturation magnetization (Ms) of Fe₃O₄ NPs. The resulting PLLA/Fe₃O₄ composite nanofibers showed paramagnetic properties with Ms directly related to the Fe₃O₄ NP concentration. The cytotoxicity of the magnetic composite nanofibers was determined using in vitro culture of osteoblasts (MC3T3-E1) in extracts and co-culture on nanofibrous matrixes. The PLLA/Fe₃O₄ composite nanofibers did not show significant cytotoxicity in comparison with pure PLLA nanofibers. On the contrary, they demonstrated enhanced effects on cell attachment and proliferation with Fe₃O₄ NP incorporation. The results suggested that this modified chemical co-precipitation method might be a universal way to produce magnetic biodegradable polyester substrates containing well-dispersed Fe₃O₄ NPs. This new strategy opens an opportunity to fabricate various kinds of magnetic polymeric substrates for bone tissue regeneration.     

Formation of bone-like apatite on poly(L-lactide) to improve osteoblast-like compatibility in vitro and in vivo

The biomimetic apatite coating process was adopted to modify poly(L-lactide) (PLLA) surfaces with osteoblasts-like cell compatibility. The apatite coating was formed on the pre-hydrolyzed PLLA film and scaffold surfaces by incubating in simulated body fluid (SBF). Scanning electron microscopy and energy dispersive X-ray analyzer were utilized to characterize the composition and the structure of the apatite coating. The cytocompatibility of the modified PLLA films was investigated by testing osteoblast-like attachment, proliferation, alkaline phosphatase (ALP) activity, and cell cycle. Subsequently, the modified PLLA scaffolds were co-cultured with the osteoblasts-like in vitro and subcutaneously implanted into nude mice. The experimental results showed that the formed apatite had a nano-sized particle and matrix configuration. The surface modification of PLLA with apatite coating significantly promoted osteoblast-like compatibility. After a four-week culture in vivo, no significant inflammatory signs were observed in the implanted regions and osteoblast-like congeries with bone-like structure began to form in the scaffolds. The positive results of this study suggest a good way to produce desirable PLLA biomaterials for bone tissue engineering.     

Biocompatibility of intraosseously implanted predegraded poly(lactide): an animal study

During degradation of high molecular weight as-polymerized poly(l-lactide) (PLLA) late complications such as swelling of the subcutaneously implanted bone plates have been observed in patients. However, in the same patients the intraosseously implanted PLLA screws did not give rise to any complications. To investigate a possible difference in the degradation and histological response due to the site of implantation, subcutaneous and intraosseous implantation studies are necessary. In the present study rods of PLLA and a copolymer poly(96l/4d-lactide) (PLA96) and predegraded PLLA and PLA96 were implanted intraosseously in rabbit tibiae. In vitro predegradation at elevated temperatures was used to obtain an enhanced degradation rate to simulate long-term physiological degradation. Animals were sacrificed after a postoperative period varying from 2 to 26 weeks. The predegraded PLLA and PLA96 implants showed a high rate of fragmentation and internalization of particles by macrophages. In the medullary cavity large fields of macrophages and, with the predegraded PLA96 implants, haemorrhages and infiltrates were noted, but no osteolytic changes were observed in the cortical bone. It can be concluded that, based on the disintegration of the implants and the accompanying histological reaction, a reaction similar to that found with subcutaneous implants in former studies had occurred. This indicates that not only the site of implantation, subcutaneous or intraosseous, but a number of other factors will affect the degradation process and the corresponding histological response.     

Nucleation and crystal growth kinetics of poly(l-lactic acid) with self-assembled DBS nanofibrils

The effect of 1,3:2,4-dibenzylidene-d-sorbitol (DBS) on the crystallization behaviors of poly(l-lactic acid) (PLLA) was examined in this study. A small amount (≤4 wt%) of DBS altered the crystallization rate and regime transition temperature of PLLA. First, the addition of DBS and the formation of self-assembled DBS nanofibrils both increased the nucleation rate of PLLA. Second, the curves of the spherulitic growth rate versus the crystallization temperature of PLLA were discontinuous and did not show the typical bell-shaped behavior for all samples. We found that the change in crystal structures (α′-to-α) affected the regime transition temperatures, which led to the discontinuity. The regime transition (regime II–III) temperatures of PLLA slightly decreased as the DBS amounts were increased. This indicates that the more regular structure (regime II) of PLLA formed at lower temperatures when more DBS was added. In addition, the spherulitic growth rate of PLLA was found to be mainly influenced by the fold surface free energy. When the DBS amounts were increased, the increase in the fold surface free energy decreased the growth rate of PLLA. Nonetheless, the Avrami exponent, n, was not significantly changed because the spherulitic growth geometry and nucleation mechanism of PLLA were basically the same. The Avrami plot also shows that the secondary crystallization began earlier due to the formation of DBS nanofibrils for the samples containing higher DBS amounts.     

Mineralization of poly(l-lactic acid)-based foams induced by cellulose nanofibrils

Cellulose nanofibrils (CNFs) were blended with poly(l-lactic acid) (PLLA) to produce CNFs/PLLA composite solid foams. The dispersed CNFs’ phase was partially embedded in the PLLA matrix. The CNFs not only reduced the water contact angle of the composite, but also induced the formation of hydroxyapatite (HA) on the walls of its inner pores. After incubation for 7 days in 3× simulated body fluid, a large number of HA particles were formed throughout the CNFs/PLLA composite foams. HA particles have diameters ranging from 200 nm to 2 μm and a Ca/P ratio of 1.42. The spatial distribution of calcium and phosphorus elements was uniform. A porosity of approximately 92 % was achieved after mineralization of the CNFs/PLLA composite foams. The mass of HA grown over CNFs/PLLA foams increased faster than in the case of PLLA foams. The ternary polymeric foams have potential applications in tissue engineering.