Nonporous Scaffolds Research Articles

Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing Applications.

Electrospun fiber scaffolds have a huge potential for the successful treatment of infected wounds based on their unique properties. Although several studies report novel drug-loaded electrospun fiber-based biomaterials, many of these do not provide information on their interactions with eukaryotic and bacterial cells. The main aim of this study was to develop antibacterial drug-loaded porous biocompatible polycaprolactone (PCL) fiber scaffolds mimicking the native extracellular matrix for wound healing purposes. Mechanical property evaluation and different biorelevant tests were conducted in order to understand the structure–activity relationships and reveal how the surface porosity of fibers and the fiber diameter affect the scaffold interactions with the living bacterial and eukaryotic fibroblast cells. Cell migration and proliferation assays and antibiofilm assays enabled us to enlighten the biocompatibility and safety of fiber scaffolds and their suitability to be used as scaffolds for the treatment of infected wounds. Here, we report that porous PCL microfiber scaffolds obtained using electrospinning at high relative humidity served as the best surfaces for fibroblast attachment and growth compared to the nonporous microfiber or nonporous nanofiber PCL scaffolds. Porous chloramphenicol-loaded microfiber scaffolds were more elastic compared to nonporous scaffolds and had the highest antibiofilm activity. The results indicate that in addition to the fiber diameter and fiber scaffold porosity, the single-fiber surface porosity and its effect on drug release, mechanical properties, cell viability, and antibiofilm activity need to be understood when developing antibacterial biocompatible scaffolds for wound healing applications. We show that pores on single fibers within an electrospun scaffold, in addition to nano- and microscale diameter of the fibers, change the living cell–fiber interactions affecting the antibiofilm efficacy and biocompatibility of the scaffolds for the local treatment of wounds.

Growth and Differentiation of Dental Stem Cells of Apical Papilla on Polycaprolactone Scaffolds.

Biodegradable scaffolds are useful tools in the field of tissue engineering and regenerative medicine. The aim of this study was to test the potential of the human stem cells of apical papilla (SCAP) to attach, proliferate and differentiate on a polycaprolactone (PCL)-based scaffolds. SCAP were extracted from the root apical papillae of freshly extracted immature premolar teeth by using enzymatic digestion. Porous PCL scaffolds were fabricated using particle leaching method and NaCl or mannitol as porogens. SCAP of passage 3 were seeded on non-porous and porous PCL scaffolds for up to 14days. For control, cells were cultured on glass coverslips. Picogreen DNA quantification was used to assay for cell proliferation. Cell differentiation and development of calcification nodules were examined using scanning electron microscopy and alizarin red staining. SCAP showed a comparable attachment, growth and proliferation patterns on PCL scaffolds and coverslips. Cell proliferation was enhanced on mannitol scaffolds at all time points. Calcification nodules were detected in all PCL scaffolds while it was not present on glass coverslips. These nodules were detected on NaCl-scaffolds by day 7 and on mannitol and non-porous scaffolds by day 14. In conclusion, SCAP were able to attach, proliferate and differentiate on PCL scaffolds without using any inductive media, indicating their potential application for dental tissue regeneration.

Acoustic characterization of 3D printed micro-structured scaffolds for tissue engineering

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate (PEGDA) hydrogel scaffolds, as a widely used biomaterial, with different geometric channels (hexagonal and square) were explored using a pulse echo technique. The measured values of attenuation and speed of sound were found to be within the range of reported values for soft tissues making PEGDA scaffolds a suitable candidate for cartilage tissue engineering. We also showed that these properties as well as Young’s modulus can be controlled and adjusted to desired values close to biological tissues by varying the 3D printing parameters. Furthermore, our 5-day proliferation as well as three-week chondrogenic differentiation results revealed that cell growth and tissue formation depend on the geometrical features of the 3D-printed scaffolds as well. Cell adhesion and proliferation greatly improved for scaffolds with square and hexagonal pore geometries compared to nonporous scaffolds. Scaffolds with square pores were determined to be the optimal for hMSC growth as well as chondrogenic differentiation. In addition to cell number, total collagen and Glycosaminoglycan (GAG) synthesis were 35% and 31% higher, respectively, for the square pattern compared to the hydrogel scaffolds with hexagonal pore shapes.

Biodegradable composite scaffolds of poly(lactic‐co‐glycolic acid) 85:15 and nano‐hydroxyapatite with acidic microclimate controlling additive

The aim of this study was to prepare and characterize biodegradable scaffolds of poly(lactic‐co‐glycolic acid) (PLGA 85:15) and nano‐hydroxyapatite (HA) containing magnesium carbonate (MC) as acidic microclimate controlling additive. Porous and nonporous scaffolds of PLGA 85:15 and nano‐HA containing MC were prepared by solvent casting and particulate leaching method. Scaffolds were degraded at pH 7.4 phosphate buffer for 90 days. At regular intervals, pH of the degradation medium, release of degradation products, mass loss, water sorption, and tensile strength were measured. The results showed that the pH of the degradation medium significantly decreased with control specimens, whereas the scaffolds with MC did not show significant changes. In addition, acidic monomer release from the scaffolds with MC was found to be less, whereas the water sorption was found to be significantly higher. The tensile strength of the scaffold was found to be dependent on the concentration of HA and porosity. These results indicate that incorporation of MC to PLGA 85:15 scaffolds reduces the acidic microclimate with changes in mass loss, water sorption, and strength properties. POLYM. COMPOS., 38:1175–1182, 2017. © 2015 Society of Plastics Engineers

Improving surface and transport properties of macroporous hydrogels for bone regeneration.

Hydrogels have been frequently considered as suitable materials for hard tissue engineering as mineralized extracellular matrix analogue. However, major lacks in bone-substitution still concern the mimicking of native microenvironment for promoting cell differentiation into osteogenic way. Here, we propose the study of mineralized macroporous hydrogels (mMHs) made of poly(ethylenglycol)diacrylate fabricated by the combination of ultraviolet photopolymerization/salt leaching technique and treated by osteopromotive medium. We demonstrate that peculiar morphological and chemical features of mMH are crucial to create a reservoir system able to efficiently recruit environmental signals to cells. In particular, mass transport mechanisms are regulated by the coupling of a Knudsen-type diffusion within the void space of the pores with a standard diffusion mechanism through the pores walls. Meanwhile, the deposition of hydrophilic mineral phases onto the pore surface further affects transport mechanisms, in view of their capability to establish interactions with water molecules and to exert mechanical constrains on the swelling of the hydrogel network, thus promoting slower diffusion kinetics. These properties concur to influence in vitro human mesenchymal stem cells activities: macropore architecture of the hydrogel-like network positively affects cell recognition as compared to nonporous scaffolds, while osteopromotive treatment mainly allows to guide differentiation in osteogenic way as proved by staining of in vitro formed Ca-rich mineral deposits (i.e., alizarin red) and expression via reverse transcription-polymerase chain reaction of main bone markers. Hence, mMH is promising to develop three-dimensional scaffolds as experimental model to study in vitro cell events during bone regeneration.

Microporous Dermal-Like Electrospun Scaffolds Promote Accelerated Skin Regeneration

The goal of this study was to synthesize skin substitutes that blend native extracellular matrix (ECM) molecules with synthetic polymers which have favorable mechanical properties. To this end, scaffolds were electrospun from collagen I (col) and poly(ɛ-caprolactone) (PCL), and then pores were introduced mechanically to promote fibroblast infiltration, and subsequent filling of the pores with ECM. A 70:30 col/PCL ratio was determined to provide optimal support for dermal fibroblast growth, and a pore diameter, 160 μm, was identified that enabled fibroblasts to infiltrate and fill pores with native matrix molecules, including fibronectin and collagen I. Mechanical testing of 70:30 col/PCL scaffolds with 160 μm pores revealed a tensile strength of 1.4 MPa, and the scaffolds also exhibited a low rate of contraction (<19%). Upon implantation, scaffolds should support epidermal regeneration; we, therefore, evaluated keratinocyte growth on fibroblast-embedded scaffolds with matrix-filled pores. Keratinocytes formed a stratified layer on the surface of fibroblast-remodeled scaffolds, and staining for cytokeratin 10 revealed terminally differentiated keratinocytes at the apical surface. When implanted, 70:30 col/PCL scaffolds degraded within 3-4 weeks, an optimal time frame for degradation in vivo. Finally, 70:30 col/PCL scaffolds with or without 160 μm pores were implanted into full-thickness critical-sized skin defects. Relative to nonporous scaffolds or sham wounds, scaffolds with 160 μm pores induced accelerated wound closure, and stimulated regeneration of healthy dermal tissue, evidenced by a more normal-appearing matrix architecture, blood vessel in-growth, and hair follicle development. Collectively, these results suggest that microporous electrospun scaffolds are effective substrates for skin regeneration.

In vitro and in vivo degradation profile of aliphatic polyesters subjected to electron beam sterilization

Degradation characteristics in response to electron beam sterilization of designed and biodegradable aliphatic polyester scaffolds are relevant for clinically successful synthetic graft tissue regeneration. Scaffold degradation in vitro and in vivo were documented and correlated to the macroscopic structure and chemical design of the original polymer. The materials tested were of inherently diverse hydrophobicity and crystallinity: poly( l-lactide) (poly(LLA)) and random copolymers from l-lactide and ε-caprolactone or 1,5-dioxepan-2-one, fabricated into porous and non-porous scaffolds. After sterilization, the samples underwent hydrolysis in vitro for up to a year. In vivo, scaffolds were surgically implanted into rat calvarial defects and retrieved for analysis after 28 and 91 days. In vitro, poly( l-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) samples degraded most rapidly during hydrolysis, due to the pronounced chain-shortening reaction caused by the sterilization. This was indicated by the rapid decrease in both mass and molecular weight of poly(LLA-co-DXO). Poly( l-lactide-co-ε-caprolactone) (poly(LLA-co-CL)) samples were also strongly affected by sterilization, but mass loss was more gradual; molecular weight decreased rapidly during hydrolysis. Least affected by sterilization were the poly(LLA) samples, which subsequently showed low mass loss rate and molecular weight decrease during hydrolysis. Mechanical stability varied greatly: poly(LLA-co-CL) withstood mechanical testing for up to 182 days, while poly(LLA) and poly(LLA-co-DXO) samples quickly became too brittle. Poly(LLA-co-DXO) samples unexpectedly degraded more rapidly in vitro than in vivo. After sterilization by electron beam irradiation, the three biodegradable polymers present widely diverse degradation profiles, both in vitro and in vivo. Each exhibits the potential to be tailored to meet diverse clinical tissue engineering requirements.

Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process

Biocompatible three-dimensional (3-D) porous scaffolds are of great interest for tissue engineering applications. We here present a novel combined freeze-drying/cross-linking process to prepare porous polysaccharide-based scaffolds. This process does not require an organic solvent or porogen agent. We unexpectedly found that cross-linking of biomacromolecules such as pullulan and dextran with sodium trimetaphosphate could be performed during freeze-drying. We have demonstrated that the freeze-drying pressure modulates the degree of porosity. High freeze-drying pressure scaffolds presented pores with a mean diameter of 55 +/- 4 microm and a porosity of 33 +/- 12%, whereas low freeze-drying pressure scaffolds contained larger pores with a mean diameter of 243 +/- 14 microm and a porosity of 68 +/- 3%. Porous scaffolds of the desired shape could be easily obtained and were stable in culture medium for weeks. In vitro viable mesenchymal stem cells were found associated with porous scaffolds in higher proportions than with non-porous scaffolds. Moreover, cells penetrated deeper into scaffolds with larger pores. This novel combined freeze-drying/cross-linking processing of polysaccharides enabled the fabrication of biocompatible scaffolds with controlled porosity and architectures suitable for 3-D in vitro culture and biomedical applications.

Macroporous photocrosslinked elastomer scaffolds containing microposity: Preparation and in vitro degradation properties

The engineering of soft tissue would benefit from the development of effective biodegradable scaffolds capable of dynamic, elastic loading. For this purpose, highly porous, elastomeric scaffolds containing microporous struts were prepared using a dual porogen approach and a photocrosslinkable elastomer. The combination of paraffin microbeads distributed through a water-in-[star-poly(lactide-co-epsilon-caprolactone) triacrylate dissolved in ethyl acetate] emulsion followed by photocrosslinking generated a macroporous foam scaffold of average porosities between 90% to 93%, with an average pore diameter of 104 +/- 31 microm with struts containing micropores of 3.1 +/- 2 microm average diameter. The mechanical properties of the scaffolds were readily manipulatable by altering the molecular weight of the star-poly(lactide-co-epsilon-caprolactone) triacrylate prepolymer used. The elastomer scaffolds degraded at the same rate as nonporous polymer samples of the same molecular weight, and exhibited similar changes in mass loss, mechanical properties, and sol fraction during in vitro degradation as found with the nonporous scaffolds. The modulus and stress at break of the scaffolds decreased continuously during degradation while the strain at break remained constant. These scaffolds show potential for use in the engineering of soft tissues.

Structural variants of biodegradable polyesterurethane in vivo evoke a cellular and angiogenic response that is dictated by architecture

The aim of this study was to investigate an in vivo tissue response to a biodegradable polyesterurethane, specifically the cellular and angiogenic response evoked by varying implant architectures in a subcutaneous rabbit implant model. A synthetic biodegradable polyesterurethane was synthesized and processed into three different configurations: a non-porous film, a porous mesh and a porous membrane. Glutaraldehyde cross-linked bovine pericardium was used as a control. Sterile polyesterurethane and control samples were implanted subcutaneously in six rabbits ( n = 12). The rabbits were killed at 21 and 63 days and the implant sites were sectioned and histologically stained using haemotoxylin and eosin (H&E), Masson’s trichrome, picosirius red and immunostain CD31. The tissue–implant interface thickness was measured from the H&E slides. Stereological techniques were used to quantify the tissue reaction at each time point that included volume fraction of inflammatory cells, fibroblasts, fibrocytes, collagen and the degree of vascularization. Stereological analysis inferred that porous scaffolds with regular topography are better tolerated in vivo compared to non-porous scaffolds, while increasing scaffold porosity promotes angiogenesis and cellular infiltration. The results suggest that this biodegradable polyesterurethane is better tolerated in vivo than the control and that structural variants of biodegradable polyesterurethane in vivo evoke a cellular and angiogenic response that is dictated by architecture.

Characterization of a slowly degrading biodegradable polyesterurethane for tissue engineering scaffolds

The purpose of this research was to develop and characterize a novel, slowly degrading polyester-urethane. In this study, a polyester-urethane with a crystalline segment of poly((R)-3-hydroxybutyric acid)-diol linked by a diisocyanate to an amorphous segment of poly(epsilon-caprolactone-co-glycolide)-diol was synthesized. Porous and nonporous scaffolds were processed using electrospinning and solvent casting respectively. The morphology, pore size, and filament diameter of the mesh and film were characterized using scanning electron microscopy (SEM). The thermal properties were examined using differential scanning calorimetry (DSC). A degradation study was initiated to characterize the change in mechanical properties, molecular weight, and surface morphology over 12 months using tensile testing, gel permeation chromatography (GPC), and SEM respectively. Concomitantly, cell morphology and viability on these variants were investigated using fibroblasts. The mechanical test data indicated a gradual decrease in the ultimate tensile strength and strain to break while the modulus of elasticity remained stable. GPC data suggested a slow decrease in the molecular weight while SEM examination revealed changed surface morphologies. The in vitro studies implied that the novel polyester-urethane was not cytotoxic and that the mesh was a more favorable scaffold towards cell viability. The summation of these results suggests that this polyester-urethane has the potential for tissue engineering applications.

Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space

Stem and progenitor cells can be combined with polymer substrates to generate tissue equivalents in culture. The replacement of retinal tissue lost to disease or trauma using retinal progenitor cells (RPCs) delivered on polymer scaffolds and transplanted into the sub-retinal space of the damaged retina is a promising therapeutic strategy. Micromachining-based, ultra-thin PMMA poly(methyl methacrylate) scaffolds may provide a suitable cytoarchitectural environment for tissue engineering and transplantation to the diseased eye. Here, adhesion of RPCs to polymer, as well as migration and differentiation in the host retina were compared for PMMA scaffolds (6 microm thickness) with either smooth or porous (11 microm diameter) surface topography. RPCs were cultured under identical conditions on smooth or porous laminin-coated polymer scaffolds and transplanted into the subretinal space of C57BL/6 mice. RPCs could be cultured on both scaffolds with similar results, although transplantation with non-porous scaffolds showed limited RPC retention. Porous scaffolds demonstrated enhanced RPC adherence during transplantation and allowed for greater process outgrowth and cell migration into the host retinal layers. Integrated cells expressed the mature neuronal marker neurofilament-200 (nf-200), the glial marker glial fibrillary acidic protein (GFAP) and the retinal-specific marker recoverin. No host foreign body response was seen. In conclusion, ultra-thin film PMMA scaffolds micromachined to contain through pores retain adherent RPCs to a considerably greater extent than unmachined versions during the transplantation process and can serve as a biocompatible substrate for cell delivery in vivo.

Development and characterization of a porous micro-patterned scaffold for vascular tissue engineering applications

The fabrication of functional small diameter blood vessel analogs has implications in vascular disease treatment. Current 3D models of the medial vessel layer lack micron-scale topographical cues that have shown promise in vitro by recapitulating native vascular smooth muscle cell (VSMC) behavior. A major obstacle to fabricating 3D scaffolds is maintaining adequate nutrient diffusion to cells. We have developed and characterized porous micro-patterned poly-caprolactone (PCL) scaffolds using a novel technique that integrates soft lithography, melt molding and particulate leaching of polylactic-co-glycolic acid (PLGA) micro/nanoparticles. Scanning electron microscopy showed that PLGA-leached scaffolds have circular pores significantly smaller than the size scale of the grooved surface pattern (48 microm grooves; 5 microm deep; 12 microm spacing). Diffusion of media through PLGA-leached scaffolds was six-fold greater than through non-porous scaffolds, indicating successful introduction of through-pores into PCL by the PLGA leaching technique. VSMC alignment on micro-patterned PLGA-leached scaffolds was similar to that on micro-patterned non-porous scaffolds, indicating no loss in cellular organization on PLGA-leached scaffolds. In contrast, cells seeded on micro-patterned sodium bicarbonate-leached scaffolds remained un-aligned. The ability to micro-pattern cells on porous scaffolds may facilitate the transfer of micro-technology from simple 2D substrates to complex 3D architectures, allowing for tight control over cellular organization in fabricated tissues.