Fabrication and properties of porous scaffold of zein/PCL biocomposite for bone tissue engineering

The aim of this study was to fabricate porous scaffolds of zein/poly(ε-caprolactone) (PCL) biocomposite by solvent casting–particulate leaching method using sodium chloride particles as the porogen. Porous biocomposite scaffolds with porosity around 70% and well-interconnected network were obtained. The incorporation of zein into PCL led to the improvement of hydrophilicity as indicated by the results of water contact angle measurement. After immersion in phosphate buffered saline (PBS) in vitro for 28 days, it was observed that the degradation rate of the zein/PCL biocomposite scaffold was faster than the PCL scaffold and that the rate could be tailored by adjusting the amount of zein in the composite. The results demonstrate the potential of the zein/PCL biocomposite scaffolds to be used in tissue engineering strategies to regenerate bone defects.     

Preparation of porous biodegradable poly(lactide-co-glycolide)/ hyaluronic acid blend scaffolds: Characterization, in vitro cells culture and degradation behaviors

A series of poly(lactide-co-glycolide) (PLGA)/ hyaluronic acid (HA) blend with different HA composition were used to fabricate scaffolds successfully. The pores of the three dimensional scaffold were prepared by particle leaching and freeze drying. The pore size was about 50–200 μ m and the porosity was about 85%. The characterizations of the scaffold, such as mechanical properties, hydrophilicity and surface morphologies were determined. Mouse 3T3 fibroblast was directly seeded on the scaffolds. The cell adhesion efficiency, cell morphology observed by scanning electron microscopy (SEM) and the degradation behavior of the blend scaffold were evaluated. In summary, the results show that the adhesion efficiency of cells on the PLGA/HA blend scaffold is higher than that on the PLGA scaffold. Moreover, the incorporation of HA in PLGA not only helps to increase the cell affinity but also tends to lead the water and nutrient into the scaffold easily.     

Porous-conductive chitosan scaffolds for tissue engineering II. <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> degradation

Porous-conductive chitosan scaffolds were fabricated by blending conductive polypyrrole (PPy) particles with chitosan solution and employing an improved phase separation method. In vitro and in vivo degradation behaviors of these scaffolds were investigated. In the case of in vitro degradation, an enzymatic degradation system was employed and lysozyme was used as a working enzyme. Meanwhile, the degradation products of scaffolds, glucosamine and N-acetyl-glucosamine, were also analyzed with a HPLC method. In vivo degradation of scaffolds was performed by subcutaneously implanting these scaffolds in rat for prescheduled time intervals. In the both cases, the weight-loss of scaffolds was monitored during the whole degradation process for evaluating the degradation of scaffolds. The changes in conductivity of scaffolds afterin vitro or in vivo degradation were also measured using a four-point technique. It was observed that the pore parameters of scaffolds themselves could significantly influence the degradation behaviors of scaffolds but the PPy content in the scaffolds seemed not to impart its effect to the degradation of scaffolds. Degradation dynamics of scaffolds and conductivity measurements indicated that these scaffolds shown fairly different behaviors in their in vitro and in vivo degradation process. According to the results obtained from in vitro and in vivo degradation of scaffolds and based on some requirements of practical tissue engineering application, it was suggested that the PPy content in the scaffold should be slightly higher than 3 wt.% but lower than 6 wt.%.     

Controlled release of BSA by microsphere-incorporated PLGA scaffolds under cyclic loading

Localized delivery of bioactive molecules from porous biodegradable scaffolds is very important in advanced tissue engineering strategies, and it is necessary to study the delivery under dynamic loading which mimics the in vivo biomechanical environments. In this study, bovine serum albumin (BSA), a model of bioactive proteins, was incorporated into porous poly(l-lactide-co-glycolide) (PLGA) scaffolds by seeding BSA-loaded microspheres onto the scaffold pore wall, where the microspheres of poly(ethylene glycol)-b-poly(l-lactide) (PELA) were prepared by double emulsion technique. The in vitro release behavior of BSA from the scaffold under dynamic cyclic loading was studied in comparison with that under a static condition as well as from PELA microspheres. It was observed that the microsphere-incorporated scaffold prolonged BSA release with respect to the microspheres. The cyclic loading accelerated the release of BSA from the scaffold and the cumulative release on day 10 reached 85% of the totally encapsulated BSA. The delivery under a dynamic condition would be an initial study of in vivo localized delivery of growth factors.     

Considerations in the development of an in vitro dissolution condition for lacidipine tablets: in vivo pharmacokinetic evaluation

In this study, a new discriminative dissolution condition for lacidipine tablets was developed by the established in vitro–in vivo relationship. Series of dissolution media of phosphate buffer solution (PBS) covering the pH range of 1–7.2 and pH 6.8 PBS containing different concentrations of sodium dodecyl sulfate (SDS), were prepared and used to investigate the dissolution behavior of lacidipine tablets. There was an obvious difference in the dissolution profiles of the both brands in pH 6.8 PBS medium containing 0.1% SDS. The pharmacokinetic study of the two lacidipine tablets was carried out in the healthy beagle dogs at a single dose of 4?mg. Statistical comparison of the AUC_0–24, C_max, and T_max showed a significant difference in the two brand tablets, coinciding with the dissolution performance with pH 6.8 PBS containing 0.1% SDS. The superiority of the proposed system, pH 6.8 PBS containing 0.1% SDS, could serve as a dissolution medium for lacidipine tablets, and more important it could discriminate the in vivo pharmacokinetic behavior for different brands of products. In summary, in vivo pharmacokinetic evaluation is essential to develop an appropriate in vitro dissolution condition for oral solid dosage forms of poorly soluble drugs.     

In vitro and in vivo degradation of poly(l-lactide-co-glycolide) films and scaffolds

Poly(l-lactide-co-glycolide) (PLGA) was synthesized using a biocompatible initiator, zirconium acetylacetonate. In vitro and in vivo degradation properties of PLGA films (produced by solvent casting, 180 μm thick) and PLGA scaffolds (produced by an innovated solvent casting and particulate leaching, 3 mm thick) were evaluated. The samples were either submitted for degradation in phosphate buffered saline (PBS) at 37 °C for 30 weeks, or implanted into rat skeletal muscles for 1, 4, 12, 22 and 30 weeks. The degradation was monitored by scanning electron microscopy, atomic force microscopy, weight loss, and molecular weight changes (in vitro), and by microscopic observations of the materials’ morphology after histological staining with May-Grunwald-Giemsa (in vivo). The results show that the films in both conditions degraded much faster than the scaffolds. The scaffolds were dimensionally stable for 23 weeks, while the films lost their integrity after 7 weeks in vitro. The films’ degradation was heterogenous—degradation in their central parts was faster than in the surface and subsurface regions due to the increased concentration of the acidic degradation products inside. In the scaffolds, having much thinner pore walls, heterogenous degradation due to the autocatalytic effect was not observed.     

In vitro investigations of a novel wound dressing concept based on biodegradable polyurethane

Abstract

Non-healing and partially healing wounds are an important problem not only for the patient but also for the public health care system. Current treatment solutions are far from optimal regarding the chosen material properties as well as price and source. Biodegradable polyurethane (PUR) scaffolds have shown great promise for in vivo tissue engineering approaches, but accomplishment of the goal of scaffold degradation and new tissue formation developing in parallel has not been observed so far in skin wound repair. In this study, the mechanical properties and degradation behavior as well as the biocompatibility of a low-cost synthetic, pathogen-free, biocompatible and biodegradable extracellular matrix mimicking a PUR scaffold was evaluated in vitro. The novel PUR scaffolds were found to meet all the requirements for optimal scaffolds and wound dressings. These three-dimensional scaffolds are soft, highly porous, and form-stable and can be easily cut into any shape desired. All the material formulations investigated were found to be nontoxic. One formulation was able to be defined that supported both good fibroblast cell attachment and cell proliferation to colonize the scaffold. Tunable biodegradation velocity of the materials could be observed, and the results additionally indicated that calcium plays a crucial role in PUR degradation. Our results suggest that the PUR materials evaluated in this study are promising candidates for next-generation wound treatment systems and support the concept of using foam scaffolds for improved in vivo tissue engineering and regeneration.     

Prolonged release from PLGA/HAp scaffolds containing drug-loaded PLGA/gelatin composite microspheres

Porous scaffolds that can prolong the release of bioactive factors are urgently required in bone tissue engineering. In this study, PLGA/gelatin composite microspheres (PGMs) were carefully designed and prepared by entrapping poly(l-lactide-co-glycolide) (PLGA) microspheres (PMs) in gelatin matrix. By mixing PGMs with PLGA solution directly, drug-loaded PLGA/carbonated hydroxyapatite (HAp)/PGMs composite scaffolds were successfully fabricated. In vitro release of fluorescein isothiocyanate-dextran (FD70S) as a model drug from the scaffolds as well as PMs and PGMs was studied by immersing samples in phosphate buffered saline (pH = 7.4) at 37°C for 32 days. Compared with PMs, PGMs and PLGA/HAp/PGMs scaffolds exhibited slow and steady release behavior with constant release rate and insignificantly original burst release. The swelling of PGMs, diffusion of drugs, and degradation of polymer dominated the release behaviors synergistically. The PLGA/HAp/PGMs scaffold offers a novel option for sequential or simultaneous release of several drugs in terms of bone regeneration.     

Evaluation of in vitro degradation of PCL scaffolds fabricated via BioExtrusion – Part 2: Influence of pore size and geometry

The in vivo degradation processes by which scaffolds degrade and are replaced by neo-tissue are complex and may be influenced by many factors, including environmental conditions, material properties, porosity and 3D architecture. The present study is focused on the influence of design parameters, filament distance (FD) and lay-down pattern, on the degradation kinetics of Polycaprolactone (PCL) scaffolds obtained via BioExtrusion. Through the variation of design parameters it was possible to obtain two groups of scaffolds with distinct pore geometry and size. The in vitro degradation was performed in simulated body fluid (SBF) and in phosphate buffer solution (PBS) for six months. Our results highlight a more complex degradation pattern of the scaffolds in SBF than in PBS, probably related to a mineral deposition. Significant statistical differences in weight loss values at month 6, allowed us to conclude that degradation kinetics of PCL scaffolds is strongly influenced by the pore size.     

Fabrication of a reticular poly(lactide-co-glycolide) cylindrical scaffold for the in vitro development of microvascular networks

Abstract

The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.     

多孔Nano-dHA/PLA/BCP复合支架的制备及性能研究

为改善常规的多孔聚乳酸/双相钙磷陶瓷(PLA/BCP)支架表面亲水性不佳及降解时呈酸性等不足,采用马弗炉烧结制备的BCP多孔支架浸入纳米缺钙羟基磷灰石/聚乳酸(nano-dHA/PLA)混悬液后,真空干燥得到多孔纳米缺钙羟基磷灰石/聚乳酸/双相钙磷陶瓷(nano-dHA/PLA/BCP)复合支架,利用万能测试机测试支架抗压强度,阿基米德法测定支架孔隙率,扫描电子显微镜(SEM)观察支架表面形貌,并对其保水率和体外降解过程中pH值的变化情况等进行了研究. 结果表明：多孔nano dHA/PLA/BCP复合支架表面粗糙,保水率和强度均有较大提高,在磷酸盐缓冲液（PBS）浸泡过程中pH值下降较慢,在模拟体液（SBF）中浸泡1个月后发现有较多的类骨磷灰石形成.     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

Processing and degradation behavior of porous magnesium scaffold for biomedical applications

Porous magnesium has the potential to be used as degradable bone scaffolds. In this study, porous magnesium scaffolds were fabricated through powder metallurgy route utilizing spherical naphthalene particle as porogen. Porogen was removed at 120?°C for 24?h followed by sintering at 550?°C for 2?h in argon atmosphere. Micro-computed tomography (micro CT) results indicated that scaffolds have interconnected porous structure with an equivalent pore diameter of nearly 60?µm. Compressive strength of the scaffolds was found in the range of 24?±?4.54?MPa to 184?±?9.9?MPa and decreased with increasing porogen content. In vitro degradation study in phosphate buffered saline (PBS) showed that scaffold degradation behavior was governed by its porosity content. Our results indicate that modulating the porogen content we can tailor the mechanical and degradation behavior of the Mg scaffolds to the application need.     

Poly(lactide-co-glycolide) encapsulated hydroxyapatite microspheres for sustained release of doxycycline

The purpose of this study was to prepare a poly(lactide-co-glycolide) (PLGA) encapsulated hydroxyapatite microspheres (HAP-MSs) as injectable depot for sustained delivery of Doxycycline (Doxy). Doxy loaded HAP-MSs (Doxy-HAP-MSs) were encapsulated with PLGA by solid-in-oil-in-water (S/O/W) emulsion-solvent evaporation technique, the effects of the PLGA used (various intrinsic viscosity and LA/GA ratio) and ratio of PLGA/HAP-MSs on the formation of Doxy-HAP-MSs and in vitro release of Doxy were studied. The results showed that sustained drug release without obvious burst was obtained by using PLGA encapsulated HAP-MSs as the carrier, also the drug release rate could be tailored by changing the ratio of PLGA/HAP-MSs, or PLGA of various intrinsic viscosities or LA/GA ratio. Lower ratio of PLGA/HAP-MSs corresponded faster Doxy release, e.g. for the microspheres of PLGA/HAP-MSs ratio of 8 and 0.25, the in vitro Doxy release percents at the end of 7days were about 23% and 76%, respectively. Higher hydrophilicity (higher ratio of GA to LA) and lower molecular weight of PLGA corresponded to higher Doxy release rates. For in vivo release study, PLGA encapsulated HAP-MSs were subcutaneously injected to the back of mice, and the results showed good correlation between the in vivo and in vitro drug release. Meanwhile, the plasma Doxy levels after subcutaneous administration of PLGA encapsulated Doxy-HAP-MSs were relatively lower and steady compared to that of the un-encapsulated microspheres. In conclusion, PLGA encapsulated HAP-MSs may be a potential vehicle for the sustained delivery of Doxy.     

In vitro and in vivo evaluation of electrospun PCL/PMMA fibrous scaffolds for bone regeneration

Abstract

Scaffolds were fabricated by electrospinning using polycaprolactone (PCL) blended with poly(methyl methacrylate) (PMMA) in ratios of 10/0, 7/3, 5/5 and 3/7. The PCL/PMMA ratio affected the fiber diameter, contact angle, tensile strength and biological in vitro and in vivo properties of the scaffolds, and the 7/3 ratio resulted in a higher mechanical strength than 5/5 and 3/7. In vitro cytotoxicity and proliferation of MG-63 osteoblast cells on these blended scaffolds were examined by MTT assay, and it was found that PCL/PMMA blends are suitable for osteoblast cell proliferation. Confocal images and expression of proliferating cell nuclear antigen confirmed the good proliferation and expression of cells on the 7/3 PCL/PMMA fibrous scaffolds. In vivo bone formation was examined using rat models, and bone formation was observed on the 7/3 PCL/PMMA scaffold within 2 months. In vitro and in vivo results suggest that 7/3 PCL/PMMA scaffolds can be used for bone tissue regeneration.     

PLGA组织工程支架材料在SBF中的降解性能和生物矿化性能

本文采用pH值测量、特性粘度、失重、DSC和电子探针的研究方法,研究了PLGA组织工程支架在模拟体液中的降解性能和生物矿化性能。研究发现随着在SBF中浸泡时间的增长,PLGA支架材料的分子量不断下降;浸泡在SBF中的PLGA组织工程支架材料的重量由沉积进程和降解进程共同决定;DSC测试显示,浸泡在SBF中的PLGA组织工程支架材料的羟基乙酸单元(GA)相对于乳酸单元(LA)更易降解;电子探针测试显示,浸泡在SBF中的PLGA组织工程支架材料表面有磷酸盐沉积物产生。     

In vitro investigations of a novel wound dressing concept based on biodegradable polyurethane

Non-healing and partially healing wounds are an important problem not only for the patient but also for the public health care system. Current treatment solutions are far from optimal regarding the chosen material properties as well as price and source. Biodegradable polyurethane (PUR) scaffolds have shown great promise for in vivo tissue engineering approaches, but accomplishment of the goal of scaffold degradation and new tissue formation developing in parallel has not been observed so far in skin wound repair. In this study, the mechanical properties and degradation behavior as well as the biocompatibility of a low-cost synthetic, pathogen-free, biocompatible and biodegradable extracellular matrix mimicking a PUR scaffold was evaluated in vitro. The novel PUR scaffolds were found to meet all the requirements for optimal scaffolds and wound dressings. These three-dimensional scaffolds are soft, highly porous, and form-stable and can be easily cut into any shape desired. All the material formulations investigated were found to be nontoxic. One formulation was able to be defined that supported both good fibroblast cell attachment and cell proliferation to colonize the scaffold. Tunable biodegradation velocity of the materials could be observed, and the results additionally indicated that calcium plays a crucial role in PUR degradation. Our results suggest that the PUR materials evaluated in this study are promising candidates for next-generation wound treatment systems and support the concept of using foam scaffolds for improved in vivo tissue engineering and regeneration.     

Fabrication,characterization and in vitro drug release behavior of electrospun PLGA/chitosan nanofibrous scaffold

In this study both aligned and randomly oriented poly(d,l-lactide-co-glycolide) (PLGA)/chitosan nanofibrous scaffold have been prepared by electrospinning. The ratio of PLGA to chitosan was adjusted to get smooth nanofiber surface. Morphological characterization using scanning electron microscopy showed that the aligned nanofiber diameter distribution obtained by electrospinning of polymer blend increased with the increase of chitosan content which was similar to that of randomly oriented nanofibers. The release characteristic of model drug fenbufen (FBF) from the FBF-loaded aligned and randomly oriented PLGA and PLGA/chitosan nanofibrous scaffolds was investigated. The drug release rate increased with the increase of chitosan content because the addition of chitosan enhanced the hydrophilicity of the PLGA/chitosan composite scaffold. Moreover, for the aligned PLGA/chitosan nanofibrous scaffold the release rate was lower than that of randomly oriented PLGA/chitosan nanofibrous scaffold, which indicated that the nanofiber arrangement would influence the release behavior. In addition, crosslinking in glutaraldehyde vapor would decrease the burst release of FBF from FBF-loaded PLGA/chitosan nanofibrous scaffold with a PLGA/chitosan ratio less than 9/1, which would be beneficial for drug release.     

Vibrational and thermal study on the in vitro and in vivo degradation of a poly(lactic acid)-based bioabsorbable periodontal membrane

Fourier transform Raman (FT-Raman), attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectra and differential scanning calorimetry (DSC) measurements were performed on a poly(lactic acid)-based biodegradable periodontal membrane in order to study its in vitro and in vivo degradation mechanism and kinetics. For this purpose, the hydrolitic in vitro degradation of the membrane was investigated in two aqueous media: saline phosphate buffer (SPB, pH=7.4) and 0.01 M NaOH solution. Moreover, a membrane implanted in vivo for four weeks for treatment of contiguous vertical bony defects, was examined. Vibrational and thermal measurements show that the membrane has a prevalently amorphous structure and is composed of low molecular weight polymeric chains. The degradation is faster in NaOH solution than in SPB and occurs heterogeneously without any significative increase in crystallinity. The DSC and spectroscopic measurements are discussed in comparison with the trend of % weight loss and show a progressive decrease in molecular weight. Regarding the Raman analysis, the I₈₇₅/I₁₄₅₂ intensity ratio was identified as a marker of the degree of degradation. Regarding the in vivo degradation, the presence, spectroscopically revealed, of a biological component entrapped in the membrane proves the good integration of the membrane with the surrounding tissues. The membrane seems to degrade faster in vivo than in vitro. A comparison with the degradation mechanism and kinetics of a periodontal membrane previously studied, Vicryl® periodontal mesh, is made.     

Effect of biomimetic 3D environment of an injectable polymeric scaffold on MG-63 osteoblastic-cell response

Solid PLGA microspheres were fabricated and characterized in terms of their in vitro degradation behaviour. Microsphere scaffolds were then modified covalently by P-15 (GTPGPQGIAGQRGVV) to obtain a 3D bioactive collagen surrogate matrix for bone filling applications. These scaffolds were characterized for surface topography, hydrophilicity and evaluated for their effect on osteoblastic activity of MG-63 cell line vis-a-vis 2D monolayer culture.AFM and contact angle experiments indicated enhanced nano-level roughness and hydrophilicity on P-15 modification. Modified scaffolds showed enhanced cell attachment, proliferation, extracellular matrix formation, mineralization and collagen type-I expression when compared to unmodified microspheres, prerequisite for bone filling applications. On long term in vitro cell culture, however, decreased cell viability was observed which may be attributed to the acidic microenvironment generated due to polymer degradation and reduction in nutrient diffusion through the copious ECM formed in 3D scaffolds. Though a higher cell count could be obtained in 2D monolayer cell culture, it was overshadowed by weak cell attachment, poor phenotypic characteristics, decreased cell viability and low mineralization levels, over 28 day cell culture studies.Results indicate that P-15 modified microsphere scaffolds may provide a natural, biomimetic 3D environment and may be successfully exploited for non-invasive bone filling applications.