PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Crosslinked collagen–gelatin–hyaluronic acid biomimetic film for cornea tissue engineering applications

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA = 6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43 × 10^? 6 cm²/s and 7.97 × 10^? 7 cm²/s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering.     

Preparation and comparative characterization of keratin–chitosan and keratin–gelatin composite scaffolds for tissue engineering applications

We report fabrication of three dimensional scaffolds with well interconnected matrix of high porosity using keratin, chitosan and gelatin for tissue engineering and other biomedical applications. Scaffolds were fabricated using porous Keratin–Gelatin (KG), Keratin–Chitosan (KC) composites. The morphology of both KG and KC was investigated using SEM. The scaffolds showed high porosity with interconnected pores in the range of 20–100 μm. They were further tested by FTIR, DSC, CD, tensile strength measurement, water uptake and swelling behavior. In vitro cell adhesion and cell proliferation tests were carried out to study the biocompatibility behavior and their application as an artificial skin substitute. Both KG and KC composite scaffolds showed similar properties and patterns for cell proliferation. Due to rapid degradation of gelatin in KG, we found that it has limited application as compared to KC scaffold. We conclude that KC scaffold owing to its slow degradation and antibacterial properties would be a better substrate for tissue engineering and other biomedical application.     

Fabrication of porous poly(L-lactide) (PLLA) scaffolds for tissue engineering using liquid–liquid phase separation and freeze extraction

PLLA scaffolds were successfully fabricated using liquid–liquid phase separation with freeze extraction techniques. The effects of different processing conditions, such as method of cooling (direct quenching and pre-quenching), freezing temperature (−80°C and −196°C) and polymer concentration (3, 5 and 7 wt%) were investigated in relations to the scaffold morphology. SEM micrographs of scaffolds showed interconnected porous network with pore size ranging from 20 to 60 μm. The scaffolds had porosity values ranging from 80 to 90%. Changes to the interconnected network, porosity and pore size were observed when the method of cooling and polymer concentration was changed. Direct quenching to −80°C gave a more porous interconnected microstructure with uniform pore size compared to samples prepared using pre-quenching method. Larger pores were observed for samples quenched at −80°C compared to −196°C. Scaffolds prepared using direct quenching to −196°C had higher elastic modulus and compressive stress compared to those quenched to −80°C. The compressive elastic modulus ranged from 4 to 7 MPa and compressive stress at 10% strain was from 0.13 to 0.18 MPa.     

Fabrication and evaluation of biomimetic scaffolds by using collagen–alginate fibrillar gels for potential tissue engineering applications

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.     

Highly porous gelatin–silica hybrid scaffolds with textured surfaces using new direct foaming/freezing technique

Highly porous gelatin–silica hybrid scaffolds with high porosity, large pores and large interconnections, as well as tailored surface textures were produced using a newly developed direct foaming/freezing. Two different types of precursors as the silica source, 3-glycidoxyproyltrimethoxysilane (denoted as “GS”) and sol–gel derived silica (denoted as “SS”), were used for producing the porous GLA–GS and GLA–GS–SS hybrid scaffolds. In this method, air bubbles could be vigorously incorporated into the GLA–GS and GLA–GS–SS mixtures and then stabilized by rapid freezing of the foamed mixtures at −70 °C. Both the porous GLA–GS and GLA–GS–SS hybrid scaffolds produced herein had a highly porous structure (porosity > 90 vol%, pore size = 200–500 μm, interconnection size = 100–200 μm) with a uniform distribution of the silica phase in the gelatin matrix. In addition, surface textures with a rugged morphology could be created after immersion of the porous GLA–GS and GLA–GS–SS hybrid scaffolds in ethanol at −20 °C for 24 h. The porous GLA–GS and GLA–GS–SS hybrid scaffolds showed much higher mechanical properties than the porous GLA scaffold, while preserving excellent in vitro biocompatibility, demonstrating potential application as the bone scaffold.     

Preparation and characterization of bimodal porous poly(γ-benzyl-L-glutamate) scaffolds for bone tissue engineering

An ideal scaffold in bone tissue-engineering strategy should provide biomimetic extracellular matrix-like architecture and biological properties. Poly(γ-benzyl-L-glutamate) (PBLG) has been a popular model polypeptide for various potential biomedical applications due to its good biocompatibility and biodegradability. This study developed novel bimodal porous PBLG polypeptide scaffolds via a combination of biotemplating method and in situ ring-opening polymerization of γ-benzyl-L-gIutamate N-carboxyanhydride (BLG-NCA). The PBLG scaffolds were characterized by proton nuclear magnetic resonance spectroscopy, X-ray diffraction, differential scanning calorimetry, scanning electron microscope (SEM) and mechanical test. The results showed that the semi-crystalline PBLG scaffolds exhibited an anisotropic porous structure composed of honeycomb-like channels (100–200 μm in diameter) and micropores (5–20 μm), with a very high porosity of 97.4 ± 1.6%. The compressive modulus and glass transition temperature were 402.8 ± 20.6 kPa and 20.2 °C, respectively. The in vitro biocompatibility evaluation with MC3T3-E1 cells using SEM, fluorescent staining and MTT assay revealed that the PBLG scaffolds had good biocompatibility and favored cell attachment, spread and proliferation. Therefore, the bimodal porous polypeptide scaffolds are promising for bone tissue engineering.     

Biomimetic design and fabrication of porous chitosan–gelatin liver scaffolds with hierarchical channel network

The presence of a hierarchical channel network in tissue engineering scaffold is essential to construct metabolically demanding liver tissue with thick and complex structures. In this research, chitosan–gelatin (C/G) scaffolds with fine three-dimensional channels were fabricated using indirect solid freeform fabrication and freeze-drying techniques. Fabrication processes were studied to create predesigned hierarchical channel network inside C/G scaffolds and achieve desired porous structure. Static in-vitro cell culture test showed that HepG2 cells attached on both micro-pores and micro-channels in C/G scaffolds successfully. HepG2 proliferated at much higher rates on C/G scaffolds with channel network, compared with those without channels. This approach demonstrated a promising way to engineer liver scaffolds with hierarchical channel network, and may lead to the development of thick and complex liver tissue equivalent in the future.     

Electrospinning of poly (ε-caprolactone-co-lactide)/Pluronic blended scaffolds for skin tissue engineering

For skin tissue engineering, an ideal scaffold should mimic the natural extracellular matrix of the native skin. In this study, we reported a novel elastic sub-micron fiber scaffold blending poly (ε-caprolactone-co-lactide) (PLCL) and Pluronic at different ratios by electrospinning. PLCL and Pluronic were co-electrospun with the ratio of 100/0, 99/1, 95/5, 90/10, 85/15, and 75/25. These scaffolds were evaluated in terms of fiber morphology, mechanical properties, and hydrophilicity for the purpose of culturing adipose-derived stem cells (ADSCs). Cell attachment and proliferation on the scaffolds were also evaluated to demonstrate the potential of serving as a skin graft. The results indicated that all of the electrospun fibers possessed smooth surface textures and interconnected porous structures with the average diameter ranging from approximately 750–1140 nm. The higher tensile strength was observed in 95/5 and 90/10 PLCL/Pluronic blended membranes, while further incorporation of Pluronic almost has no effect on tensile strength. The water contact angle was 85° for scaffold with the ratio of 99/1, while 0° for 90/10, 85/15, and 75/25. In addition, the elevation of Pluronic content in composition resulted in a corresponding increase in swelling behavior. Compared with PLCL, the better cell adhesion and proliferation potential of ADSCs was exhibited on all PLCL/Pluronic blended scaffolds. ADSCs on the blended scaffolds were highly elongated and well integrated with the surrounding fibers, indicating the good cytocompatibility of PLCL/Pluronic scaffolds. Thus, these blended scaffolds have the potentially high application prospect in the field of skin tissue engineering.     

Bioglass®-based scaffolds with carbon nanotube coating for bone tissue engineering

Highly porous 45S5 Bioglass^®-based foam scaffolds were coated with multi-walled carbon nanotubes (CNT) by electrophoretic deposition (EPD) technique. By placing the scaffolds in between the two electrodes of the EPD cell, a CNT coating of up to 1 μm thickness was achieved on the surface throughout the whole three dimensional (3D) matrix. A 0.5 wt% CNT aqueous suspension was used and EPD was carried out at 2.8 V for 10 mins. The compression strength of this CNT/Bioglass^® composite was measured to be 0.70 MPa. Moreover the increased electrical conductivity of the composite with CNT coating was confirmed. The scaffolds have the potential for applications in bone tissue engineering due to the high bioactivity, nano-roughness in 3D and electrical conductivity provided by the addition of CNT.     

Glycolic acid-functionalized chitosan–Co3O4–Fe3O4 hybrid magnetic nanoparticles-based nanohybrid scaffolds for drug-delivery and tissue engineering

In the present work, Co₃O₄ was prepared by hydrothermal process, which is further used for the synthesis of Co₃O₄–Fe₃O₄ hybrid nanoparticles. The formation of Co₃O₄–Fe₃O₄ nanoparticles was investigated by transmission electron microscopy and physical property measurement system. In the next step, the drug-loaded novel nanohybrid porous scaffold based on chitosan-g-glycolic acid and Co₃O₄–Fe₃O₄ nanoparticle was prepared by freeze drying technique. The grafting of glycolic acid on chitosan drug loading in porous scaffold was characterized by Fourier transform infrared spectroscopy. The nanohybrid scaffolds were found to be stable regardless of the pH of the medium and play an important role in cell adhesion, proliferation, and migration. Co₃O₄–Fe₃O₄ hybrid nanoparticles' reinforcement was found to control the drug (cyclophosphamide) release rate in phosphate buffer saline solution (pH 7.4). Therefore, Co₃O₄–Fe₃O₄ hybrid nanoparticles are viable additives for formulating sustained drug delivery systems and could be applied in the field of biomaterials.     

Sol–gel method to fabricate CaP scaffolds by robocasting for tissue engineering

Highly porous calcium phosphate (CaP) scaffolds for bone-tissue engineering were fabricated by combining a robocasting process with a sol–gel synthesis that mixed Calcium Nitrate Tetrahydrate and Triethyl Phosphite precursors in an aqueous medium. The resulting gels were used to print scaffolds by robocasting without the use of binder to increase the viscosity of the paste. X-ray diffraction analysis confirmed that the process yielded hydroxyapatite and β-tricalcium phosphate biphasic composite powders. Thus, the scaffold composition after crystallization of the amorphous structure could be easily modified by varying the initial Ca/P ratio during synthesis. The compressive strengths of the scaffolds are ~6 MPa, which is in the range of human cancellous bone (2–12 MPa). These highly porous scaffolds (~73 vol% porosity) are composed of macro-pores of ~260 μm in size; such porosity is expected to enable bone ingrowth into the scaffold for bone repair applications. The chemistry, porosity, and surface topography of such scaffolds can also be modified by the process parameters to favor bone formation. The studied sol–gel process can be used to coat these scaffolds by dip-coating, which induces a significant enhancement of mechanical properties. This can adjust scaffold properties such as composition and surface morphology, which consequently may improve their performances.     

Novel mineralized heparin–gelatin nanoparticles for potential application in tissue engineering of bone

Nanoparticles (NPs) were prepared from succinylated gelatin (s-GL) cross-linked with aldehyde heparin (a-HEP) and used subsequently as a nano-template for the mineralization of hydroxyapatite (HAP). Gelatin was functionalized with succinyl groups that made it soluble at room temperature. Heparin was oxidized to generate aldehyde groups and then used as a cross-linker that can react with s-GL to form NPs via Schiff’s base linkage. The polymer concentrations, feed molar ratios and pH conditions were varied to fabricate NPs suspension. NPs were obtained with a spheroid shape of an average size of 196 nm at pH 2.5 and 202 nm at pH 7.4. These NPs had a positive zeta potential of 7.3 ± 3.0 mV and a narrow distribution with PDI 0.123 at pH 2.5, while they had a negative zeta potential of ?2.6 ± 0.3 mV and formed aggregates (PDI 0.257) at pH 7.4. The NPs prepared at pH 2.5 with a mean particle size of 196 nm were further used for mineralization studies. The mineralization process was mediated by solution without calcination at 37 °C. The HAP formed on NPs was analyzed by Fourier transform infrared spectroscopy and X-ray diffraction. HAP coated s-GL/a-HEP NPs developed in this study may be used in future as osteoinductive fillers enhancing the mechanical properties of injectable hydrogel or use as potential multifunctional device for nanotherapeutic approaches.     

Supermacroprous chitosan–agarose–gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering

The study focuses on the synthesis of a novel polymeric scaffold having good porosity and mechanical characteristics synthesized by using natural polymers and their optimization for application in cartilage tissue engineering. The scaffolds were synthesized via cryogelation technology using an optimized ratio of the polymer solutions (chitosan, agarose and gelatin) and cross-linker followed by the incubation at sub-zero temperature (−12°C). Microstructure examination of the chitosan–agarose–gelatine (CAG) cryogels was done using scanning electron microscopy (SEM) and fluorescent microscopy. Mechanical analysis, such as the unconfined compression test, demonstrated that cryogels with varying chitosan concentrations, i.e. 0.5–1% have a high compression modulus. In addition, fatigue tests revealed that scaffolds are suitable for bioreactor studies where gels are subjected to continuous cyclic strain. In order to confirm the stability, cryogels were subjected to high frequency (5 Hz) with 30 per cent compression of their original length up to 1 × 10⁵ cycles, gels did not show any significant changes in their mass and dimensions during the experiment. These cryogels have exhibited degradation capacity under aseptic conditions. CAG cryogels showed good cell adhesion of primary goat chondrocytes examined by SEM. Cytotoxicity of the material was checked by MTT assay and results confirmed the biocompatibility of the material. In vivo biocompatibility of the scaffolds was checked by the implantation of the scaffolds in laboratory animals. These results suggest the potential of CAG cryogels as a good three-dimensional scaffold for cartilage tissue engineering.     

A porous scaffold for bone tissue engineering/45S5 Bioglass<Superscript>®</Superscript> derived porous scaffolds for co-culturing osteoblasts and endothelial cells

One of the major factors in the therapeutic success of bone tissue engineered scaffolds is the ability of the construct to vascularise post implantation. One of the approaches for improving vascularisation within scaffolds has been to co-culture human umbilical vein endothelial cells (HUVECS) with human osteoblasts (HOBS), which may then promote vascularisation and facilitate tissue regeneration. However, in order to mimic a natural physiological niche it is vital that the scaffold is able to support and promote the proliferation of both cell types and thus become a viable tissue engineered construct. In this study we report the development of a porous bioactive glass–ceramic construct and examine the interaction with human umbilical vein endothelial cells (HUVEC’s) and human osteoblast-like cell both in mono and co-culture. The study clearly demonstrated that the scaffolds were able to support both endothelial and human osteoblast cell proliferation both in mono and co-culture. A comparison of the proliferation response of HUVEC and HOB in mono-culture on the test scaffolds and the commercial porous hydroxyapatite was assessed over a 28 day period (4, 7, 14, 21 and 28 days), using alamar Blue^TM assay. Proliferation of HOB cells seeded in the scaffolds was consistently shown to be above those observed on commercial HA scaffolds.     

Fabrication of three-dimensional poly(ε-caprolactone) scaffolds with hierarchical pore structures for tissue engineering

The physical properties of tissue engineering scaffolds such as microstructures play important roles in controlling cellular behaviors and neotissue formation. Among them, the pore size stands out as a key determinant factor. In the present study, we aimed to fabricate porous scaffolds with pre-defined hierarchical pore sizes, followed by examining cell growth in these scaffolds. This hierarchical porous microstructure was implemented via integrating different pore-generating methodologies, including salt leaching and thermal induced phase separation (TIPS). Specifically, large (L, 200–300 μm), medium (M, 40–50 μm) and small (S, < 10 μm) pores were able to be generated. As such, three kinds of porous scaffolds with a similar porosity of ~ 90% creating pores of either two (LS or MS) or three (LMS) different sizes were successfully prepared. The number fractions of different pores in these scaffolds were determined to confirm the hierarchical organization of pores. It was found that the interconnectivity varied due to the different pore structures. Besides, these scaffolds demonstrated similar compressive moduli under dry and hydrated states. The adhesion, proliferation, and spatial distribution of human fibroblasts within the scaffolds during a 14-day culture were evaluated with MTT assay and fluorescence microscopy. While all three scaffolds well supported the cell attachment and proliferation, the best cell spatial distribution inside scaffolds was achieved with LMS, implicating that such a controlled hierarchical microstructure would be advantageous in tissue engineering applications.     

Evaluation of 3D nano–macro porous bioactive glass scaffold for hard tissue engineering

Recently, nano–macro dual-porous, three-dimensional (3D) glass structures were developed for use as bioscaffolds for hard tissue regeneration, but there have been concerns regarding the interconnectivity and homogeneity of nanopores in the scaffolds, as well as the cytotoxicity of the environment deep inside due to limited fluid access. Therefore, mercury porosimetry, nitrogen absorption, and TEM have been used to characterize nanopore network of the scaffolds. In parallel, viability of MG 63 human osteosarcoma cells seeded on scaffold surface was investigated by fluorescence, confocal and electron microscopy methods. The results show that cells attach, migrate and penetrate inside the glass scaffold with high proliferation and viability rate. Additionally, scaffolds were implanted under the skin of a male New Zealand rabbit for in vivo animal test. Initial observations show the formation of new tissue with blood vessels and collagen fibers deep inside the implanted scaffolds with no obvious inflammatory reaction. Thus, the new nano–macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration.     

Nanofibrous membrane of collagen–polycaprolactone for cell growth and tissue regeneration

Nanofibrous substrates of synthetic polymers including polycaprolactone (PCL) have shown considerable potential in tissue regeneration. This paper reports the use of PCL/collagen nanofibers to improve the in vitro osteoblastic responses for the applications in bone regeneration area. Collagen and PCL were dissolved in a co-solvent, and the resulting solution was electrospun into a nanofibrous web. Nonwoven fibrous matrices were successfully produced at various compositional ratios (PCL/collagen = 1/3, 1 and 3 by weight). Although the PCL nanofiber was hydrophobic, the presence of collagen significantly improved the water affinity, such as the water contact angle and water uptake capacity. Tensile mechanical tests showed that the collagen–PCL nanofiber had a significantly higher extension rate (approximately 2.8-fold) than the PCL while maintaining the maximum tensile load in a similar range. The osteoblastic cells cultured on the collagen–PCL nanofibrous substrate showed better initial adhesion and a higher level of growth than those cultured on the PCL nanofiber. Furthermore, real-time RT-PCR revealed the expression of a series of bone-associated genes, including osteopontin, collagen type I and alkaline phosphatase. The expression of these genes was significantly higher on the collagen–PCL nanofiber than on the PCL nanofiber. When subcutaneously implanted in mouse the collagen–PCL membrane facilitated tissue cells to well penetrate into the nanofibrous structure at day 7, whilst no such cell penetration was noticed in the pure PCL nanofiber. Overall, the presence of collagen within the PCL nanofiber improves the water affinity, tensile extension rate, and the tissue cell responses, such as initial adhesion, growth, penetration and the expression of bone-associated genes. Therefore, the collagen–PCL nanofibrous membrane may have potential applications in the cell growth and bone tissue regeneration.