Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions

We present polymeric hydrogel biomaterials that are biomimetic both in their synthesis and degradation. The design of oligopeptide building blocks with dual enzymatic responsiveness allows us to create polymer networks that are formed and functionalized via enzymatic reactions and are degradable via other enzymatic reactions, both occurring under physiological conditions. The activated transglutaminase enzyme factor XIIIa was utilized for site-specific coupling of prototypical cell adhesion ligands and for simultaneous cross-linking of hydrogel networks from factor XIIIa substrate-modified multiarm poly(ethylene glycol) macromers. Ligand incorporation is nearly quantitative and thus controllable, and does not alter the network's macroscopic properties over a concentration range that elicits specific cell adhesion. Living mammalian cells can be encapsulated in the gels without any noticeable decrease in viability. The degradation of gels can be engineered to occur, for example, via cell-secreted matrix metalloproteinases, thus rendering these gels interesting for biomedical applications such as drug delivery systems or smart implants for in situ tissue engineering.     

Poly(ether urethane) networks from renewable resources as candidate biomaterials: synthesis and characterization

A series of poly(ether urethane) networks were synthesized from epoxidized methyl-oleate-based polyether polyol and 1,3-propandiol using l-lysine diisocyanate as a nontoxic coupling agent. Polyurethanes with different hard segment contents were prepared to tune the final properties of the materials. The polyurethanes were fully chemically and physically characterized, including water uptake and in vitro hydrolytic degradation measurements. The weight loss of the polyurethanes was traced, and the changes in the surface morphology with the degradation time were examined by scanning electron microscopy. The experimental results revealed that the hard segment content is the main factor that controls the physical, mechanical, and degradation properties of these polymers. The observed diversity in material properties suggests that these polyurethanes may be useful for a wide range of biomedical polymer applications.     

Polymers of malic acid and 3-alkylmalic acid as synthetic PHAs in the design of biocompatible hydrolyzable devices.

Poly(beta-malic acid) and poly(beta-3-alkylmalic acid) derivatives, as synthetic polyhydroxyalkanoates (PHAs), present several advantages as macromolecular materials for temporary biomedical applications. Indeed, such polymers, which can be synthesized through different chemical and biological routes, have cleavable ester bonds in their backbone for hydrolytic degradation, stereogenic centres in the monomers units for controlling the macromolecular structure. bioassimilable or non-toxic repeating units and lateral chemical functions which can be adapted to specific requirements. The strategy for building such complex architectures, with one or several specific pendant groups, is based on the anionic ring-opening polymerization or copolymerization of the large family of malolactonic and 3-alkylmalolactonic acid esters. Because we are able to control the monomer synthesis and the polymerization step, we have been able to prepare different degradable materials for the biomedical field, such as: degradable associating networks made up by the association of random copolyesters containing a small percentage of hydrophobic moieties and beta-cyclodextrin copolymers; degradable macromolecular micelles constituted by degradable amphiphilic block copolymers of poly(beta-malic acid) as hydrophilic segments and poly(beta-alkylmalic acid alkyl esters) as hydrophobic blocks; and degradable nanoparticles made up by hydrophobic poly(beta-malic acid alkyl esters) derivatives. We have also prepared a terpolymer which exhibits growth factor-like properties in vivo. Finally, poly(beta-malic acid) has been used as an additive in the preparation of peritoneal dialysis bags.     

Hydrolytic and enzymatic degradation of nanoparticles based on amphiphilic poly(gamma-glutamic acid)-graft-L-phenylalanine copolymers

Amphiphilic graft copolymers consisting of poly(gamma-glutamic acid) (gamma-PGA) as the hydrophilic backbone and L-phenylalanine ethylester (L-PAE) as the hydrophobic side chain were synthesized by grafting L-PAE to gamma-PGA. The nanoparticles were prepared by a precipitation method, and about 200 nm-sized nanoparticles were obtained due to their amphiphilic properties. The hydrolytic and enzymatic degradation of these gamma-PGA nanoparticles was studied by gel permeation chromatography (GPC), scanning electron microscopy (SEM), dynamic light scattering (DLS) and (1)H NMR measurements. The hydrolysis ratio of gamma-PGA and these hydrophobic derivatives was found to decrease upon increasing the hydrophobicity of the gamma-PGA derivates. The pH had an effect on the hydrolytic degradation of the polymer. The hydrolysis of the polymer could be accelerated by alkaline conditions. The degradation of the gamma-PGA backbone by gamma-glutamyl transpeptidase (gamma-GTP) resulted in a dramatic change in nanoparticle morphology. With increasing time, the gamma-PGA nanoparticles began to decrease in size and finally disappeared completely. Moreover, the gamma-PGA nanoparticles were degraded by four different enzymes (Pronase E, protease, cathepsin B and lipase) with different degradation patterns. The enzymatic degradation of the nanoparticles occurred via the hydrolysis of gamma-PGA as the main chain and L-PAE as the side chain. In the case of the enzymatic degradation of gamma-PGA nanoparticles with Pronase E, the size of the nanoparticles increased during the initial degradation stage and decreased gradually when the degradation time was extended. Nanoparticles composed of biodegradable amphiphilic gamma-PGA with reactive function groups can undergo further modification and are expected to have a variety of potential pharmaceutical and biomedical applications, such as drug and vaccine carriers.     

Poly(L-lactide-co-glycolide) thin films can act as autologous cell carriers for skin tissue engineering

Degradable aliphatic polyesters such as polylactides, polyglycolides and their copolymers are used in several biomedical and pharmaceutical applications. We analyzed the influence of poly(L-lactide-co-glycolide) (PLGA) thin films on the adhesion, proliferation, motility and differentiation of primary human skin keratinocytes and fibroblasts in the context of their potential use as cell carriers for skin tissue engineering. We did not observe visible differences in the morphology, focal contact appearance, or actin cytoskeleton organization of skin cells cultured on PLGA films compared to those cultured under control conditions. Moreover, we did not detect biologically significant differences in proliferative activity, migration parameters, level of differentiation, or expression of vinculin when the cells were cultured on PLGA films and tissue culture polystyrene. Our results indicate that PLGA films do not affect the basic functions of primary human skin keratinocytes and fibroblasts and thus show acceptable biocompatibility in vitro, paving the way for their use as biomaterials for skin tissue engineering.     

Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver

Porous scaffolds for tissue engineering applications based on poly(D,L-lactide)/poly(epsilon-caprolactone) compatibilized blends are described. The addition of a third polymer, namely poly( D, L-lactide-co-caprolactone) copolymer, has a profound effect on morphological properties of the blends scaffolds. In fact, the copolymer acts as compatibilizing agent and reduces the dimension of the dispersed phase of an order of magnitude. Such effect is function of the polymer composition. The efficiency of scaffolds obtained with poly( D, L-lactide) based blends containing 30% by weight of poly(epsilon-caprolactone) as dispersed phase toward hepatocytes has been tested by several biological assays and we found that they are able to promote a perfect adhesion, proliferation and growth of cells. Moreover, the addition of the copolymer significantly improves the biomedical performance of the scaffold.     

Development of a biostable replacement for PEGDA hydrogels

The exceptional tunability of poly(ethylene glycol) (PEG) hydrogel chemical, mechanical, and biological properties enables their successful use in a wide range of biomedical applications. Although PEG diacrylate (PEGDA) hydrogels are often used as nondegradable controls in short-term in vitro studies, it is widely acknowledged that the hydrolytically labile esters formed upon acrylation of the PEG diol make them susceptible to slow degradation in vivo. A PEG hydrogel system that maintains the desirable properties of PEGDA while improving biostability would be valuable in preventing degradation-related failure of gel-based devices in long-term in vivo applications. To this end, PEG diacrylamide (PEGDAA) hydrogels were synthesized and characterized in quantitative comparison to traditional PEGDA hydrogels. It was found that PEGDAA hydrogel modulus and swelling can be tuned over a similar range and to comparable degrees as PEGDA hydrogels with changes in macromer molecular weight and concentration. Additionally, PEGDAA cytocompatibility, low cell adhesion, and capacity for incorporation of bioactivity were analogous to that of PEGDA. In vitro hydrolytic degradation studies showed that the amide-based PEGDAA had significantly increased biostability relative to PEGDA. Overall, these findings indicate that PEGDAA hydrogels are a suitable replacement for PEGDA hydrogels with enhanced hydrolytic resistance. In addition, these studies provide a quantitative measure of the hydrolytic degradation rate of PEGDA hydrogels which was previously lacking in the literature.     

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide-maleic anhydride copolymer (PEO-MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis-Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.     

Mathematical model of the role of degradation on matrix development in hydrogel scaffold

Despite tremendous advances in the field of tissue engineering, a number of obstacles are still hindering its successful translation to the clinic. One of these challenges has been to design cell-laden scaffolds that can provide an appropriate environment for cells to successfully synthesize new tissue while providing a mechanical support that can resist physiological loads at the early stage of in situ implementation. A solution to this problem has been to balance tissue growth and scaffold degradation by creating new hydrogel systems that possess both hydrolytic and enzymatic degradation behaviors. Very little is known, however, about the complex behavior of these systems, emphasizing the need for a rigorous mathematical approach that can eventually assist and guide experimental advances. This paper introduces a mathematical and numerical formulation based on mixture theory, to describe the degradation, swelling, and transport of extracellular matrix (ECM) molecules released by cartilage cells (chondrocytes) within a hydrogel scaffold. The model particularly investigates the relative roles of hydrolytic and enzymatic degradations on ECM diffusion and their impacts on two important outcomes: the extent of ECM transport (and deposition) and the evolution of the scaffold’s mechanical integrity. Numerical results based on finite element show that if properly tuned, enzymatic degradation differs from hydrolytic degradation in that it can create a degradation front that is key to maintaining scaffold stiffness while allowing ECM deposition. These results therefore suggest a hydrogel design that could enable successful in situ cartilage tissue engineering.     

PGS支架的合成、特性及在生物医学中应用的研究进展

聚癸二酸甘油酯(PGS)是一种生物可降解的高分子聚合弹性体,因其良好的性能,在许多生物医学研究中应用广泛。PGS支架的机械性能与机体软组织相似,依从性好,降解时以表面侵蚀的方式降解,不伴有膨胀或变形,周围组织炎症反应、纤维变性轻,与多种细胞相容性好。基于PGS良好的性能,主要应用于软组织替代和软组织工程,比如心肌、血管、神经、软骨、视网膜、鼓膜,另外也有用于药物转运载体、组织粘附材料的研究。     

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco‐friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA‐chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF‐chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491–500, 2016     

Advances in biomedical applications of pectin gels

Pectin, due to its simple and cytocompatible gelling mechanism, has been recently exploited for different biomedical applications including drug delivery, gene delivery, wound healing and tissue engineering. Recent studies involving pectin for the biomedical field are reviewed, with the aim to capture the state of art on current research about pectin gels for biomedical applications, moving outside the traditional fields of application such as the food industry or pharmaceutics. Pectin structure, sources and extraction procedures have been discussed focussing on the properties of the polysaccharide that can be tuned to optimize the gels for a desired application and possess a fundamental role in application of pectin in the biomedical field.     

Polycaprolactone scaffolds fabricated with an advanced electrohydrodynamic direct-printing method for bone tissue regeneration

Electrohydrodynamic (EHD) direct writing has been used in diverse microelectromechanical systems and various supplemental methods for biotechnology and electronics. In this work, we expanded the use of EHD-induced direct writing to fabricate 3D biomedical scaffolds designed as porous structures for bone tissue engineering. To prepare the scaffolds, we modified a grounded target used in conventional EHD direct printing using a poly(ethylene oxide) solution bath, elastically cushioning the plotted struts to prevent crumbling. The fabricated scaffolds were assessed for not only physical properties including surface roughness and water uptake ability but also biological capabilities by culturing osteoblast-like cells (MG63) for the EHD-plotted polycaprolactone (PCL) scaffold. The EHD-scaffolds showed significantly roughened surface and enhanced water-absorption ability (400% increase) compared with the pure rapid-prototyped PCL. The results of cell viability, alkaline phosphatase activity, and mineralization analyses showed significantly enhanced biological properties of the scaffold (20 times the cell viability and 6 times the mineralization) compared with the scaffolds fabricated using RP technology. Because of the results, the modified EHD direct-writing process can be a promising method for fabricating 3D biomedical scaffolds in tissue engineering.     

Poly(caprolactone-co-oxo-crown ether)-based poly(urethane)urea for soft tissue engineering applications

Random copolymers of epsilon-caprolactone and 2-oxo-12-crown-4 ether, poly(CL-co-OC), were used as soft segments in the synthesis of a set of poly(urethane)urea thermoplastic elastomers. With increasing OC content, the soft segment crystallinity decreased, which influenced the mechanical properties: strain induced crystallization disappeared upon the introduction of OC into poly(CL). The material therefore became weaker, however, without a reduction in strain at break. All polymers showed mechanical properties that are suitable for soft tissue engineering. Degradation studies of poly(CL-co-OC) copolymers revealed a higher intrinsic rate of hydrolysis as compared to poly(CL). When at least two neighboring OC units were present in the soft segment, a jump in the intrinsic hydrolysis rate was observed. From this study we deduced an ideal OC:CL ratio for the thermoplastic elastomer soft segments for soft tissue engineering applications. An in vitro degradation study of these poly(urethane)urea showed an increased weight loss. Combined with the enhanced hydrophilicity and reduced crystallinity, we are confident that this will indeed lead to an increased degradation rate in vivo.     

Polyhydroxyalkanoate (PHA)/inorganic phase composites for tissue engineering applications

Polyhydroxyalkanoates are emerging as a class of biodegradable polymers for applications in tissue engineering. Members of the polyhydroxyalkanoates family encompass a wide variety of materials, from hard and brittle materials to soft and elastomeric. Over the years, efforts have been made to extend the group of polyhydroxyalkanoates and to investigate their use in numerous biomedical applications, such as sutures, cardiovascular patches, wound dressings, guided tissue repair/regeneration devices, and tissue engineering scaffolds. Along with the development of polyhydroxyalkanoates, researchers have looked into the possibility of designing composites in combination with inorganic phases to further improve the mechanical properties, rate of degradation, and also impart bioactivity. Poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) are some of the polymers which have been studied extensively to fabricate composites in combination with hydroxyapatite, bioactive glass, and glass-ceramic fillers or coatings. This paper reviews international research carried out toward development of polyhydroxyalkanoates/inorganic phase composites in terms of systems investigated, microstructures, properties achieved, and applications, with special focus on tissue engineering scaffolds. A comparison between different composite systems developed in the past few years is presented. The paper also addresses the prospect of potential further development of polyhydroxyalkanoates/inorganic phase composites with optimized microstructure and properties for improved tissue engineering scaffolds.     

Self-assembly and photo-cross-linking of eight-armed PEG-PTMC star block copolymers

Eight-armed poly(ethylene glycol)-poly(trimethylene carbonate) star block copolymers (PEG-(PTMC)(8)) linked by a carbamate group between the PEG core and the PTMC blocks were synthesized by the metal-free, HCl-catalyzed ring-opening polymerization of trimethylene carbonate using an amine-terminated eight-armed star PEG in dichloromethane. Although dye solubilization experiments, nuclear magnetic resonance spectroscopy, and dynamic light scattering clearly indicated the presence of aggregates in aqueous dispersions of the copolymers, no physical gelation was observed up to high concentrations. PEG-(PTMC(9))(8) was end-group-functionalized using acryloyl chloride and photopolymerized in the presence of Irgacure 2959. When dilute aqueous dispersions of PEG-(PTMC(9))(8)-Acr were UV irradiated, chemically cross-linked PEG-PTMC nanoparticles were obtained, whereas irradiation of more concentrated PEG-(PTMC(9))(8)-Acr dispersions resulted in the formation of photo-cross-linked hydrogels. Their good mechanical properties and high stability against hydrolytic degradation make photo-cross-linked PEG-PTMC hydrogels interesting for biomedical applications such as matrices for tissue engineering and controlled drug delivery systems.     

Synthesis of Biodegradable and Electroactive Tetraaniline Grafted Poly(ester amide) Copolymers for Bone Tissue Engineering

Biodegradable poly(ester amide)s have recently been used as biomaterials due to their desirable chemical and biological characteristics as well as their mechanical properties, which are amendable for material processing. In this study, electroactive tetraaniline (TA) grafted poly(ester amide)s were successfully synthesized and characterized. The poly(ester amide)s-graft-tetraaniline copolymers (PEA-g-TA) exhibited good electroactivity, mechanical properties, and biodegradability. The biocompatibility of the PEA-g-TA copolymers in vitro was systematically studied, which demonstrated that they were nontoxic and led to favorable adhesion and proliferation of mouse preosteoblastic MC3T3-E1 cells. Moreover, the PEA-g-TA copolymers stimulated by pulsed electrical signal could serve to promote the differentiation of MC3T3-E1 cells compared with TCPs. Hence, the biodegradable and electroactive PEA-g-TA copolymers possessed the properties in favor of the long-time potential application in vivo (electrical stimulation directly to the desired area) as bone repair scaffold materials in tissue engineering.     

Polyhedral oligomeric silsesquioxane (POSS) suppresses enzymatic degradation of PCL-based polyurethanes

In this Article, we studied the enzymatic hydrolytic biodegradation behavior of a novel multiblock thermoplastic polyurethane (TPU) system, which incorporates polyhedral oligomeric silsesquioxane (POSS) into linear biodegradable thermoplastic polyurethanes containing poly(ε-caproactone) (PCL) and polyethylene glycol (PEG) blocks. The biodegradation behavior of POSS-PCL-PEG TPUs was characterized by proton nuclear magnetic resonance spectroscopy ((1)H NMR), differential scanning calorimetry (DSC), tensile tests, scanning electron microscopy (SEM), and wavelength dispersive X-ray spectrometry (WDS) after enduring 22-day accelerated enzymatic hydrolytic degradation tests. POSS incorporation significantly suppressed in vitro enzymatic hydrolytic degradation of PCL-PEG-based multiblock TPUs by a surface passivation mechanism. WDS observations revealed that the covalently bonded POSS moieties developed a near-continuous and robust POSS-layer after initial degradation, which prevented ester bonds of PCL from enzymatic attack, thereby inhibiting further degradation. These striking results provide a new strategy to fabricate the polyester-based biostable thermoplastic polyurethanes (TPUs) of potential use in long-term surgical implants.     

Smart hydrogels based on semi-interpenetrating polymeric networks of collagen-polyurethane-alginate for soft/hard tissue healing,drug delivery devices,and anticancer therapies

In this work, hydrogels based on semi-interpenetrating polymeric networks (semi-IPN) based on collagen-polyurethane-alginate were studied physicochemically and from different approaches for biomedical application. It was determined that the matrices in the hydrogel state are crosslinked by the formation of urea and amide bonds between the biopolymer chains and the polyurethane crosslinker. The increment in alginate content (0–40 wt%) significantly increases the swelling capacity, generating semi-crystalline granular structures with improved storage modulus and resistance to thermal, hydrolytic, and proteolytic degradation. The in vitro bioactivity results indicated that the composition of these novel hydrogels stimulates the metabolic activity of monocytes and fibroblasts, benefiting their proliferation; while in cancer cell lines, it was determined that the composition of these biomaterials decreases the metabolic activity of breast cancer cells after 48 h of stimulation, and for colon cancer cells their metabolic activity decreases after 72 h of contact for the hydrogel with 40 wt% alginate. The matrices show a behavior of multidose release of ketorolac, and a higher concentration of analgesic is released in the semi-IPN matrix. The inhibition capacity of Escherichia coli is higher if the polysaccharide concentration is low (10 wt%). The in vitro wound closure test (scratch test) results indicate that the hydrogel with 20 wt% alginate shows an improvement in wound closure at 15 days of contact. Finally, the bioactivity of mineralization was evaluated to demonstrate that these hydrogels can induce the formation of carbonated apatite on their surface. The engineered hydrogels show biomedical multifunctionality and they could be applied in soft and hard tissue healing strategies, anticancer therapies, and drug release devices.     

Synthesis of biodegradable and electroactive multiblock polylactide and aniline pentamer copolymer for tissue engineering applications

To obtain one biodegradable and electroactive polymer as the scaffold for tissue engineering, the multiblock copolymer PLAAP was designed and synthesized with the condensation polymerization of hydroxyl-capped poly( l-lactide) (PLA) and carboxyl-capped aniline pentamer (AP). The PLAAP copolymer exhibited excellent electroactivity, solubility, and biodegradability. At the same time, as one scaffold material, PLAAP copolymer possesses certain mechanical properties with the tensile strength of 3 MPa, tensile Young 's modulus of 32 MPa, and breaking elongation rate of 95%. We systematically studied the compatibility of PLAAP copolymer in vitro and proved that the electroactive PLAAP copolymer was innocuous, biocompatible, and helpful for the adhesion and proliferation of rat C6 cells. Moreover, the PLAAP copolymer stimulated by electrical signals was demonstrated as accelerating the differentiation of rat neuronal pheochromocytoma PC-12 cells. This biodegradable and electroactive PLAAP copolymer thus possessed the properties in favor of the long-time application in vivo as nerve repair scaffold materials in tissue engineering.