Design of tunable protein-releasing nanoapatite/hydrogel scaffolds for hard tissue engineering

Hierarchically structured porous scaffolds based on nanocrystalline carbonated hydroxyapatite reinforced hydrogels (Gellan or Agarose) have been tested as protein release matrices while evaluation their in vitro biocompatibility. The shaping method used develops under mild conditions thus allowing the incorporation of labile substances. The Bovine Serum Albumin (BSA), employed as a model protein, has been included by using two drug-inclusion strategies: during the scaffolds preparation (in situ process) or by injection of an aqueous protein solution within (ex situ process). The release studies showed a more controlled BSA delivery when the protein was incorporated during the scaffold preparation when compared to that where the protein has been loaded in a second step (ex situ process). The release patterns can also be tailored as a function of the scaffold composition (ceramic/polysaccharide ratio and nature) as well as the drying technology employed. Biocompatibility studies demonstrated that these scaffolds, regardless of the composition, allow the culture of osteoblasts on and around the material, thus supporting the potential use of these biomaterials for bone tissue engineering.     

Controlled release system for ametryn using polymer microspheres: preparation, characterization and release kinetics in water

The purpose of this work was to develop a modified release system for the herbicide ametryn by encapsulating the active substance in biodegradable polymer microparticles produced using the polymers poly(hydroxybutyrate) (PHB) or poly(hydroxybutyrate-valerate) (PHBV), in order to both improve the herbicidal action and reduce environmental toxicity. PHB or PHBV microparticles containing ametryn were prepared and the efficiencies of herbicide association and loading were evaluated, presenting similar values of approximately 40%. The microparticles were characterized by scanning electron microscopy (SEM), which showed that the average sizes of the PHB and PHBV microparticles were 5.92±0.74 μm and 5.63±0.68 μm, respectively. The ametryn release profile was modified when it was encapsulated in the microparticles, with slower and more sustained release compared to the release profile of pure ametryn. When ametryn was associated with the PHB and PHBV microparticles, the amount of herbicide released in the same period of time was significantly reduced, declining to 75% and 87%, respectively. For both types of microparticle (PHB and PHBV) the release of ametryn was by diffusion processes due to anomalous transport (governed by diffusion and relaxation of the polymer chains), which did not follow Fick's laws of diffusion. The results presented in this paper are promising, in view of the successful encapsulation of ametryn in PHB or PHBV polymer microparticles, and indications that this system may help reduce the impacts caused by the herbicide, making it an environmentally safer alternative.     

Mathematical modelling of the evolution of protein distribution within single PLGA microspheres: prediction of local concentration profiles and release kinetics

Protein release from poly(D,L-lactide-co-glycolide) (PLGA) microspheres in an aqueous environment is governed by the diffusion of the protein through an autocatalytically degrading polymeric matrix. Many attempts have been made to model the release rate of proteins from biodegrading matrices, but the transport parameters involved in the process are not fully established at the microscale level. The aim of this work was to develop a new mathematical model taking into account the temporal evolution of the radial protein distribution during release, and to provide physical insight into the relation between local transport features and microsphere degradation. The model was validated by comparing its predictions with the experimentally determined protein concentration profiles in PLGA microspheres loaded with tetramethylrhodamine-labelled bovine serum albumin (BSA-Rhod) as a model protein. Morphological studies were carried out by scanning electron microscopy (SEM), while release kinetics and time-dependent BSA-Rhod concentration profiles within the microspheres were studied by a confocal laser scanning microscopy (CLSM)-assisted technique. The model, based on a modification of Fick's second law of diffusion, could closely fit the experimental protein radial distribution profiles in the microspheres as a function of time. It is also a useful tool to ab initio design protein release devices using degrading matrices.     

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.     

A novel method for the preparation of biodegradable microspheres for protein drug delivery.

Microspheres are potential candidates for the protein drug delivery. In this work, we prepared polymer-coated starch/bovine serum albumin (BSA) microspheres using co-axial electrohydrodynamic atomization (CEHDA). First, starch solution in dimethyl sulphoxide (DMSO) was prepared and then an aqueous solution of BSA was added to it to make a starch-BSA solution. Subsequently, this solution was made to flow through the inner capillary, while the polymer, polydimethylsiloxane (PDMS), flowed through the outer capillary. On collection, filtration and subsequent drying, near-monodisperse microspheres of 5-6microm in size were obtained. The microspheres were characterized by Fourier-transform infrared (FT-IR) spectroscopy and scanning electron microscopy. Cumulative BSA release was investigated by UV spectroscopy. BSA structure and activity was preserved in the microspheres and its release in 0.01M phosphate buffered saline (PBS) was studied over a period of 8 days. There was an initial burst with 32wt% of total BSA released in 2h. Overall 75wt% of BSA was released over a 7 day period.     

Bioactivity evaluation of collagen-based scaffolds containing a series of Sr-doped melt-quench derived phosphate-based glasses

Phosphate-based glasses have been attracting attention due to their possible medical applications arising from unique dissolution characteristics in the human body leading to the possibility of new tissue regeneration. In this study, the leaching kinetics of a series of melt-quenched Sr-doped phosphate glasses are presented. Regardless of the presence of Sr, all the glasses have an initial linear and sustained release of the ions followed by a plateau. To guarantee proper nutritional support to the growing tissue during regeneration and to mimic the 3-dimensional architecture of tissues, organic scaffold systems have been developed. However, their poor mechanical strength has limited their application. To overcome this problem, cross-linkers can be used although this then limits the solubility of the materials. To succeed in dealing with such a limitation, in this paper, by freeze-drying, the aforementioned soluble melt-quenched phosphate glasses were combined as powders with collagen fibres from bovine achilles tendon to make degradable scaffolds. The scaffolds were characterized by SEM, EDX and BET. Changes to the dissolution behaviour of the glasses arising from the presence of collagen interacting with the ions leached were reported. Furthermore, the ability of the scaffolds to induce hydroxyapatite (HA) formation was evaluated: one the elaborated scaffold could grow an HA-like layer after a week in SBF. Based on the results obtained, a possible application in restorative dentistry is proposed for one or more materials.

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Evaluation of BSA protein release from hollow hydroxyapatite microspheres into PEG hydrogel

Implants that simultaneously function as an osteoconductive matrix and as a device for local drug or growth factor delivery could provide an attractive system for bone regeneration. In our previous work, we prepared hollow hydroxyapatite (abbreviated HA) microspheres with a high surface area and mesoporous shell wall and studied the release of a model protein, bovine serum albumin (BSA), from the microspheres into phosphate-buffered saline (PBS). The present work is an extension of our previous work to study the release of BSA from similar HA microspheres into a biocompatible hydrogel, poly(ethylene glycol) (PEG). BSA-loaded HA microspheres were placed in a PEG solution which was rapidly gelled using ultraviolet radiation. The BSA release rate into the PEG hydrogel, measured using a spectrophotometric method, was slower than into PBS, and it was dependent on the initial BSA loading and on the microstructure of the microsphere shell wall. A total of 35–40% of the BSA initially loaded into the microspheres was released into PEG over ~ 14 days. The results indicate that these hollow HA microspheres have promising potential as an osteoconductive device for local drug or growth factor delivery in bone regeneration and in the treatment of bone diseases.     

Towards excimer-laser-based stereolithography: a rapid process to fabricate rigid biodegradable photopolymer scaffolds

We demonstrate high-resolution photocross-linking of biodegradable poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) using UV excimer laser photocuring at 308 nm. The curing depth can be tuned in a micrometre range by adjusting the total energy dose (total fluence). Young''s moduli of the scaffolds are found to be a few gigapascal, high enough to support bone formation. The results presented here demonstrate that the proposed technique is an excellent tool for the fabrication of stiff and biocompatible structures on a micrometre scale with defined patterns of high resolution in all three spatial dimensions. Using UV laser photocuring at 308 nm will significantly improve the speed of rapid prototyping of biocompatible and biodegradable polymer scaffolds and enables its production in a few seconds, providing high lateral and horizontal resolution. This short timescale is indeed a tremendous asset that will enable a more efficient translation of technology to clinical applications. Preliminary cell tests proved that PPF : DEF scaffolds produced by excimer laser photocuring are biocompatible and, therefore, are promising candidates to be applied in tissue engineering and regenerative medicine.     

Physicochemical characterisation of novel ultra-thin biodegradable scaffolds for peripheral nerve repair

In this study, the physicochemical properties of microporous poly (ε-caprolactone) (PCL) films and a composite material made of PCL and polylactic acid (PLA) blend were tested. Fabricated by solvent casting using dichloromethane, these ultra-thin films (60 ± 5 μm in thickness) have a novel double-sided surface topography, i.e. a porous surface with pores 1–10 μm in diameter and a relatively smooth surface with nano-scaled texture. Porous surfaces were found to be associated with increased protein adsorption and the treatment of these polyester scaffolds with NaOH rendered them more hydrophilic. Differential Scanning Calorimetry (DSC) showed that the incorporation of PLA reduced the crystallinity of the original homopolymer. Chemical changes were investigated by means of Fourier Transform Infrared Spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Average surface roughness (Ra), hydrophilicity/hydrophobicity and mechanical properties of these materials were also assessed for the suitability of these materials as nerve conduits.     

蛋白质生物降解塑料的研究

概括了蛋白质生物降解塑料的研究 ,包括蛋白质塑料的降解原理 ,研究用蛋白种类 ,蛋白塑料的改性方法 ,以及蛋白塑料制样方式等 ,并对其发展前景进行了展望。     

可生物降解聚合物微球的制备及载药应用

综述了近年来合成生物降解聚合物微球的种类、制备方法及作为药物载体的应用.结果表明,这类聚合物微球具有良好的安全性、生物相容性和生物降解性,在药物缓释、控释和疾病的治疗中具有非常重要的作用和应用前景.     

A novel method for fabrication of biodegradable scaffolds with high compression moduli

It has been previously shown that, when used for meniscal reconstruction, porous copoly(L-lactide/-caprolactone) implants enhanced healing of meniscal lesions owing to their excellent adhesive properties. However, it appeared that the materials had an insufficient compression modulus to accomplish 100% fibrocartilage formation. In addition, to be used for meniscal prosthesis, the compression modulus of the porous materials should be larger than 150 kPa in order to protect the articular cartilage. A technique was developed to prepare stiff porous materials of a high molecular weight 50/50 copoly(L-lactide/-caprolactone) suitable for fibrocartilage regeneration in meniscal implants and meniscal prosthesis. Porous microspheres (50–250 m) were agglutinated in the presence of NaCl crystals (250–300 m). The microspheres were mixed with solid solvent in order to obtain a homogeneous distribution of solvent over the spheres. By changing the amount of solvent and crystals, the density and the compression modulus could be varied over a range of 0.07 g ml-1 to 0.5 g dl-1 and 40–1100 kPa, respectively.     

Zonal release of proteins within tissue engineering scaffolds

The manufacture of a scaffold for tissue engineering applications that can control the location and timing of growth factor release is described. The scaffold is formed by the sintering of poly(DL-lactic acid) (P_DLLA) microparticles, plasticized with poly(ethylene glycol) (PEG), although the method can be used for many other polymer types. The microparticles were loaded with model proteins, trypsin and horseradish peroxidase (HRP), or recombinant human bone morphogenetic protein-2 (rhBMP-2). Entrapment efficiencies above 75% were achieved using a solid-in-oil-in-water system. Controlled release of active protein was achieved for at least 30 days. Microparticles were built into protein-loaded or protein-free layers and release of the protein was restricted to zones within the scaffold. Cell response to rhBMP-2 was tuneable by changing the dose of the rhBMP-2 released by varying the ratio of protein-loaded and protein-free microparticles within scaffolds. Zonal activity of rhBMP-2 on C2C12 cells was demonstrated. The scaffolds may find utility in applications where gradients of growth factors within 3D templates are required or where zonation of tissue growth is required.     

生物可降解高分子多孔微球制备与应用的研究进展

生物可降解高分子多孔微球可以在体内降解并且无毒副作用,已经被广泛应用于药物控制释放、组织工程等领域,它们的制备及应用是近年来的研究热点。概述了生物可降解高分子多孔微球的几种制备方法及其应用领域,并对生物可降解高分子多孔微球的未来发展前景进行了展望。     

Enzyme-nanoparticle functionalization of three-dimensional protein scaffolds

Various surface modification techniques have been developed for patterning functional biomolecules in two dimensions, allowing enzymes, antibodies, and other compounds to be localized for applications in bioanalysis and bioengineering. Here, we report a strategy for extending high-resolution patterning of biomolecules to three dimensions. In this approach, three-dimensional protein scaffolds are created by a direct-write process in which multiphoton excitation promotes photochemical cross-linking of protein molecules from aqueous solution within specified volume elements. After scaffold fabrication, protein microstructures are functionalized with enzyme-gold nanoparticle conjugates via a targeting process based in part on electrostatic attraction between the low-isoelectric-point enzyme and the microstructure, fabricated from high-isoelectric-point proteins. High signal-to-background ratios (approximately 20:1) are demonstrated for fluorescent product streams created by dephosphorylation of the fluorogenic compound, fluorescein diphosphate, at microstructures decorated with alkaline phosphatase-gold nanoparticle conjugates. We also demonstrate feasibility for using such structures to quantify substrate concentrations in flowing streams with low-micromolar detection limits and to create sensor suites based on both enzyme-nanoparticle functionalization and intrinsic enzymatic activity of protein scaffolds. These topographically complex sensors and dosing sources have potential applications in microfluidics, sensor array fabrication, and real-time chemical modification of cell culture environments.     

Fabrication and characterization of chitosan microspheres agglomerated scaffolds for bone tissue engineering

In the presented paper authors describe a method for bone scaffolds fabrication. The technique is based on the agglomeration of chitosan microspheres. The fabrication process is complex and consists of a few steps: chitosan spheres extrusion, scaffold formation by compression followed by the spheres agglomeration and bonding with cross-linking agent (STPP, sodium tripolyphosphate). The described method allows manufacturing of porous materials with controllable shape, pore size distribution and their interconnectivity. In this technique 3D scaffold porosity can be regulated by altering spheres diameter. Authors studied influence of cross-linker concentrations and time of cross-linking process on the scaffold morphology, mechanical properties, enzymatic degradation rate (in the presence of lysozyme) and human osteoblasts response. Surface morphology and topography were evaluated by SEM. Porosity and pore interconnectivity were observed via μCT scanning. Mechanical tests showed that chitosan scaffolds perform compression characteristic (Young Modulus) similar to natural bone. Cytotoxicity established by XTT assay confirmed that most of the developed composite materials do not show toxic properties. Osteoblast adhesion and morphology were analyzed by SEM and optical microscopy.     

Manufacture and evaluation of bioactive and biodegradable materials and scaffolds for tissue engineering

For tissue regeneration and tissue engineering applications, a number of bioactive and biodegradable composites, either porous or non-porous, were fabricated. The newly developed materials included tricalcium phosphate reinforced polyhydroxybutyrate and its copolymer, poorly crystallized hydroxyapatite reinforced chitin, and plasma sprayed hydroxyapatite reinforced poly(L-lactic acid). It was shown that these new materials could be successfully produced using the manufacturing techniques adopted. In vitro experiments revealed that the incorporation of bioceramic particles in biodegradable polymers rendered the composites bioactive and significantly improved the ability of composites to induce the formation of bone-like apatite on their surfaces. Degradation of composite scaffolds in simulated body fluid was observed and could be due to the simultaneous degradation of polymer matrix and dissolution of bioceramic particles.     

Control of morphological properties of porous biodegradable scaffolds processed by supercritical CO2 foaming

A low cost supercritical CO₂ foaming rig with a novel design has been used to prepare fully interconnected and highly porous biodegradable scaffolds with controllable pore size and structure that can promote cancellous bone regeneration. Porous polymer scaffolds have been produced by plasticising the polymer with high pressure CO₂ and by the formation of a porous structure following the escape of CO₂ from the polymer. Although, control over pore size and structure has been previously reported as difficult with this process, the current study shows that control is possible. The effects of processing parameters such as CO₂ saturation pressure, time and temperature and depressurisation rate on the morphological properties, namely porosity, pore interconnectivity, pore size and wall thickness- of the scaffolds have been investigated. Poly(d,l)lactic acid was used as the biodegradable polymer. The surfaces and internal morphologies of the poly(d,l)lactic acid scaffolds were examined using optical microscope and micro computed tomography. Preosteoblast human bone cells were seeded on the porous scaffolds in vitro to assess cell attachment and viability. The scaffolds showed a good support for cell attachment, and maintained cell viability throughout 7 days in culture. This study demonstrated that the morphology of the porous structure can be controlled by varying the foaming conditions, allowing the porous scaffolds to be used in various tissue engineering applications.     

20-Hydroxyecdysone release from biodegradable devices: the effect of size and shape

20-Hydroxyecdysone (20-OH) is a natural compound with many demonstrated effects on the physiological functions of vertebrates, particularly increased protein synthesis. Our study sought a suitable dosage form with continuous release of the drug lasting several weeks for implantation into agricultural animals. Biodegradable microparticles and implants of poly(L-lactic) and poly(DL-lactic) acids were prepared. Oligomers of these materials were synthesized, and a method of melting the binary mixture of the oligomer and 20-OH was employed. The particles were prepared simply by grinding the solidified block of the melt and sieving. Implants were prepared by extruding the melt into silicone tubes, removing the solidified content, and cutting into cylinders of 2 mm diameter and various lengths. A new method of preparation of hollow cylinders by aspirating air into silicone tubes filled with the melt was developed. The experiments demonstrated stability of 20-OH during heat treatment. Release of the active ingredient was tested in static in vitro conditions, analogous to those at the site of implantation, and prolonged drug release was obtained with both types of implant. The hollow implants gave release rates nearest to ideal zero-order kinetics and would appear most appropriate for testing in vivo.