ABSTRACT
Recently, modifying bacterial cellulose (BC) by compositing it with other nano-biomaterials has become inevitable to achieve its desired properties in drug delivery. To address this, our study endeavors to utilize an in-situ fabrication method for the creation of a multifunctional BC/gelatin (BC/Gel) platform reinforced with carboxylic multi-walled carbon nanotubes (cMWCNTs) as a sustainable delivery model of biomolecules. Incipiently, cMWCNTs were loaded with human serum albumin (HSA) as a drug model, with an optimized nanoparticle-to-protein ratio of 1:5 and loading efficiency of 90.0 ± 1.0 % before incorporation into BC/Gel hydrogels. By comparison, nanocomposition improved the surface area and overall porosity of BC/Gel up to 58.0 ± 1.3 m2/g and 85.5 ± 1.1 %, respectively. Likewise, significant wettability of 44.0 ± 0.1° and dramatic biodegradation rate of 36.9 ± 1.2 % were other exceptionally gained attributes. Meanwhile, with a Zero-order kinetic mechanism, CNT-HSA integration facilitated the controlled release of 56.0 ± 0.9 % HSA over 7 days. Drug-loaded nanocomposites showcased >70 % viability during in vitro cellular trials using Human Foreskin Fibroblasts (HFF). Overall, BC/Gel/CNT-HSA nanocomposite exhibited favorable cell behavior, devoid of cytotoxic manifestations. Consequently, this BC-based nanocomposite scaffold implicates the premiere capability in the sustained delivery of an extended range of protein biomolecules, offering a promising therapeutic avenue for bolstering tissue regeneration.
Subject(s)
Nanocomposites , Nanotubes, Carbon , Humans , Cellulose/metabolism , Gelatin , Biocompatible Materials , Bacteria/metabolismABSTRACT
Ciliary neurotrophic factor (CNTF) promotes survival and/or differentiation of a variety of neuronal cells including retinal ganglion cells (RGCs). Delivery of CNTF requires a suitable medium capable of mediating diffusion and premature release of CNTF within the target tissue. Polymeric tissue-engineered scaffolds have been readily used as substrates for cell transplantation, expansion, and differentiation and, as carriers of cell growth factors. Their functions to CNTF release for RGC proliferation have remained so far unexplored, especially to CNTF affinity to the scaffold and subsequent RGC fate. Electrospunpoly(glycerol sebacate)/poly(ϵ-caprolactone) (PGS/PCL) biopolymer scaffolds have recently shown promising results in terms of supporting regeneration of RGC neurites. This work explores covalent immobilization of CNTF on PGS/PCL scaffold and the way immobilised CNTF mediates growth of RGC axons on the scaffold. Anex-vivothree-dimensional model of rodent optic nerve on PGS/PCL revealed that RGC explants cultured in CNTF mediated environment increased their neurite extensions after 20 d of cell culture employing neurite outgrowth measurements. The CNTF secretion on PGS/PCL scaffold was found bio-mimicking natural extracellular matrix of the cell target tissue and, consequently, has shown a potential to improve the overall efficacy of the RGC regeneration process.
Subject(s)
Ciliary Neurotrophic Factor , Retinal Ganglion Cells , Retinal Ganglion Cells/metabolism , Ciliary Neurotrophic Factor/metabolism , Axons/physiology , Neurites/metabolism , Cell Proliferation , Nerve Regeneration/physiology , Cell Survival/physiologyABSTRACT
Materials that speed up wound healing can be of great benefit to patients and healthcare providers. One-layer dressings, however, have unsatisfactory healing efficacy since it is impossible to use materials with different properties simultaneously, and drug delivery is limited by the depth of penetration. The present study utilized a multilayer wound dressing composed of bacterial cellulose (BC) hydrogel, gelatin/alginate (Gel/Alg) hydrogel, and polycaprolactone (PCL) nanofibers loaded with ciprofloxacin (CIP) to promote the healing process in vivo. The designed dressings showed significant water absorption and sufficient water vapor transmission rate (WVTR) after one week, confirming their ability to absorb wound exudate. Within the first four hours, significant amounts of CIP were released from the drug-containing dressing. Then, between hours 4 and 24, the rate decreased and plateaued on day 9. Both positive and negative bacterial strains were inhibited by the gradual release of CIP, while fibroblasts retained their normal morphology and metabolic activity. Lastly, in vivo tests demonstrated that CIP-loaded multilayer dressings could significantly speed up full-thickness wound healing during 14 days, by reducing inflammation, stimulating re-epithelialization, and enhancing skin regeneration. Our findings indicate that multilayering BC hydrogels with drug-loaded nanofibers provide a promising way to promote wound healing by utilizing all the distinctive properties of these layers.
Subject(s)
Cellulose , Wound Healing , Humans , Skin , Hydrogels/pharmacology , Bacteria , Ciprofloxacin/pharmacology , Anti-Bacterial Agents/pharmacologyABSTRACT
Various therapeutic strategies have been developed to address bone diseases caused by aging society and skeletal defects caused by trauma or accidental events. One such approach is using bone fillers, such as hydroxyapatite (HA) and bioactive glasses. Although they have provided effective osteogenesis, infection and inflammation due to the surgical procedure and uncontrolled ion release can hinder the efficiency of bone regeneration. In response to these challenges, immobilizing a neutral metal-phenolic network on the surface of osteoconductive nanoparticles would be the master key to achieving a gradual, controlled release during the mineralization period and reducing infection and inflammation through biological pathways. In this regard, a mesoporous silica nanocomposite modified by an HA precursor was synthesized to enhance bone regeneration. In addition, to improve the therapeutic effects, its surface was wrapped with a magnesium-phenolic network made from pomegranate extract, which can simultaneously produce anti-inflammatory and antibacterial effects. The obtained core-shell nanocomposite was characterized by its physicochemical properties, biocompatibility, and bioactivity. The in vitro studies revealed that the synthesized nanocomposite exhibits higher osteogenic activity than the control groups, as confirmed by alizarin red staining. Moreover, the nanocomposite maintained low toxicity as measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and increased antibacterial activity against Staphylococcus aureus and Escherichia coli compared with the control groups. Therefore, this research presents a promising strategy for bone regeneration, combining the advantages of mesoporous silica nanocomposite modified by an HA precursor with the beneficial effects of a magnesium-phenolic network.
Subject(s)
Durapatite , Magnesium , Humans , Durapatite/pharmacology , Durapatite/chemistry , Silicon Dioxide/pharmacology , Osteogenesis , Bone Regeneration , Anti-Bacterial Agents/toxicity , Anti-Bacterial Agents/chemistry , InflammationABSTRACT
In tissue engineering and regenerative medicine, nature's template is the extracellular matrix (ECM) which provides mechanical support and biochemical signals to encourage cell attachment and modulate cell behavior. Biological and biomimetic materials derived from the decellularized ECM (dECM) are successfully used in a variety of biomedical therapies both in preclinical studies and clinical applications. One of the main goals of decellularization is the elimination of cellular components from tissue samples or whole organs while maintaining their resident regulatory molecules and mechanical integrity. The coupling of decellularized bioproducts with computer-aided fabrication technologies such as three-dimensional (3D) printing, is revolutionizing tissue engineering and regenerative medicine. Following the growth of different computer-aided scaffolding methods, 3D bioprinting offers the possibility of incorporating different cells into the biomaterial formulation to produce a cell-laden structure. Suitable materials for 3D bioprinting are often referred to as bioinks and have become an important field research area. The concept of bioink, originally considered a printable hydrogel that incorporated living cells, has recently changed. That is to say, biomaterials that can be printed and subsequently seeded with cells after printing, do not qualify as a bioink. However, the 3D printing of biomaterial inks and bioinks of dECM for cartilage regeneration is still burgeoning. Cartilage possesses uniquely complex mechanical properties that are integral to tissue function and can be attributed to the ECM network. Hence, using a printed dECM scaffold could be an encouraging approach to engineering cartilage while preserving its depth-dependent ECM structure. This paper provides an overview of the composition and structure of cartilage ECM and 3D printing and bioprinting of dECM-related scaffolds, emphasizing cartilage function.
Subject(s)
Biocompatible Materials , Bioprinting , Biocompatible Materials/chemistry , Tissue Scaffolds/chemistry , Ink , Tissue Engineering/methods , Extracellular Matrix/chemistry , Cartilage , Printing, Three-Dimensional , Bioprinting/methodsABSTRACT
3D printing has recently emerged as an innovative fabrication method to construct critical-sized and patient-specific bone scaffolds. The ability to control the bulk geometry of scaffolds in both macro and micro-scales distinguishes this technology from other fabrication methods. In this study, bone tissue-specific scaffolds with different pore geometries were printed from polylactic acid (PLA) filaments at three given infill densities ranging from 20 to 30%. A hybrid hydrogel made of synthetic biphasic calcium phosphate (BCP) and collagen was applied to coat 3D printed well-structured triangular samples with 30% infill density. The coating process changed the surface texture, increased the average strand diameter and average pore size, and decreased the open porosity of samples, all of which increased the mechanical strength of biomimetic-coated scaffolds. According to matrix mineralization staining and osteo-related gene expression, the coating of scaffolds significantly facilitates metabolic activity and osteogenic differentiation of dental pulp-derived mesenchymal stem cells (DPSCs). Taken together, these results indicated that the biomimetic coating is a highly promising approach that could be taken into consideration in the design of a porous scaffold for bone tissue engineering.
Subject(s)
Biomimetics , Osteogenesis , Humans , Tissue Scaffolds , Tissue Engineering/methods , Polyesters , Printing, Three-Dimensional , PorosityABSTRACT
Tissue-specific extracellular matrix (ECM) plays a critical role in cell survival and homeostasis, which are particularly essential for directing differentiation of different complex tissues such as retina. However, ECM maintenance should be considered to design an effective therapeutic strategy for retina regeneration. To achieve this, cell sheet engineering has emerged as a growing approach to closely reconstruct basal membrane of cells through a scaffold-free manner. Several irreversible sight-threatening diseases are characterized by the dysfunction and lose of retinal pigment epithelium (RPE), leading to vision loss and eventually total blindness in patients. According to impressive developments in achievement of RPE from human embryonic stem cells (hESCs), we obtained RPE cells without any extrinsic factors in a co-culture system, and cultured them on a temporary alginate hydrogel substrate. Subsequently, Arg-Gly-Asp (RGD) peptide was superficially immobilized on the upper layer of hydrogel to improve cell attachment before harvesting sheet layer. RPE cell sheet layer was released by treating pre-seeded hydrogels with sodium citrate as a calcium chelating agent and characterized in both in vitro and in vivo models. RPE sheets formed tight junction and expressed high levels of retina structural markers such as ZO-1, Bestrophin and Collagen type IV. One week after in vivo transplantation of RPE sheet, cells survived in the subretinal space, indicating that our harvesting method is non-invasive. To sum up, we introduced a unique scaffold-free method for RPE cell sheet engineering, which can find potential use for future therapeutic purposes.
Subject(s)
Alginates , Retinal Pigment Epithelium , Alginates/chemistry , Cell Differentiation , Humans , Hydrogels/chemistry , Oligopeptides/metabolism , Retinal Pigment Epithelium/metabolismABSTRACT
The stem cell-based retinal ganglion cells (RGCs) replacement therapy offers a potential to restore vision in progressive optic neuropathies including glaucoma by replacing degenerated RGCs and by simulating axonal regeneration. Injured optic nerve axons do not regenerate owing to the limited intrinsic capacity of the neurons and the inhibitory environment at the injury site. Polymeric tissue scaffolds are able to modulate the physical environment while providing structural support for transplanted cells, however, their application specific to the RGC generation has been far from conclusive. The successful generation of clinically safe and functional RGCs that can appropriately integrate into the hosts' retinas still remain largely unresolved. Our study reports on a process that enables generation of RGCs from human embryonic stem cells (hESCs) that is simple, straightforward and repeatable and, investigates the influence of the aligned poly(glycerol sebacate) (PGS)/poly(ε-caprolactone) (PCL) scaffold on this differentiation process. Our findings demonstrate that PGS/PCL scaffold promotes differentiation of hESCs into RGC-like cells possibly by the simulation of cell active environmental signalling and, facilitates the growth of RGCs neurites along their lengths. STATEMENT OF SIGNIFICANCE: Glaucoma can lead to the degeneration of retinal ganglion cells (RGCs), with consequential vision loss. RGCs are incapable of self-renewal, replacement of diseased RGCs with healthy cells has been a goal to restore vision in glaucoma patients. In this regard, stem cell RGC replacement therapy has been shown to improve vision in animal models of glaucoma, which could be facilitated by using tissue-engineered polymeric scaffolds. In this study, we generated homogenous stem cell-derived RGCs via a straightforward differentiation protocol and evaluated the effects of PGS/PCL scaffold on RGCs differentiation and growth of RGCs neurites. Our study contributes to the knowledge on how biomaterial scaffolds are able to support the regeneration of RGC neurites (i.e., axons or dendrites) as a part of a possible future clinical therapy for the treatment of glaucoma.
Subject(s)
Human Embryonic Stem Cells , Retinal Ganglion Cells , Animals , Axons , Cell Differentiation , Humans , Optic NerveABSTRACT
Cartilage regeneration using biomaterial-guided delivery systems presents improved therapeutic efficacy of the biomolecules while minimizing side effects. Here, our hypothesis was to design a multilayer scaffold of chitosan (CS) hydrogel and polycaprolactone (PCL) mat to enhance the mechanical properties, integrity and stability of CS, especially for subsequent in vivo transplantation. After conjugation of the Kartogenin (KGN) into this structure, its gradual release can promote chondrogenesis of mesenchymal stem cells (MSCs). Initially, a thin electrospun PCL layer was sandwiched between two CS hydrogels. Subsequently, KGN was superficially immobilized onto the CS matrix. The successful conjugation was confirmed by scanning electron microscopy (SEM) and infrared spectroscopy. These novel KGN-conjugated scaffolds possessed lower swelling and higher compressive modulus and showed gradual release of KGN in longer retention times. Immunofluorescent and histological staining represented more cells located in lacunae as well as more Coll2 and Sox9 positive cells on KGN-conjugated scaffolds. Gene expression analysis also revealed that SOX9, COLL2 and ACAN expression levels were higher in the presence of KGN, while COLLX expression was down-regulated, indicating a hypertrophy phenomenon with synergistic effect of TGF-ß. This multilayer structure not only facilitates the effective treatment, but also provides a proper mechanical structure for cartilage engineering.
Subject(s)
Cartilage/physiology , Chitosan , Chondrocytes/metabolism , Delayed-Action Preparations , Polyesters , Regeneration/drug effects , Anilides/chemistry , Anilides/pharmacokinetics , Anilides/pharmacology , Cells, Cultured , Chitosan/chemistry , Chitosan/pharmacology , Humans , Phthalic Acids/chemistry , Phthalic Acids/pharmacokinetics , Phthalic Acids/pharmacology , Polyesters/chemistry , Polyesters/pharmacologyABSTRACT
Bioengineered scaffolds composed of synthetic materials and extracellular matrix (ECM) components can offer a tissue-specific microenvironment capable of regulating cells to regenerate the structure and function of the native cartilage. Here, given the potential preservation of biomechanical and biochemical cues found in the native cartilage, particulate decellularized ECM (DC-ECM) was utilized for immobilization on the surface of nanofibrous scaffolds. Afterward, the chondro-inductive potential and ectopic cartilage formation after subcutaneous implantation of bioengineered DC-ECM scaffolds were investigated in mice model. Eight weeks post-implantation, no growth of considerable inflammatory response and neovascularization was observed in histological images of bioengineered DC-ECM scaffolds. Pre-seeded bioengineered scaffolds with human adipose-derived stem cells exhibited high levels of chondro-induction capability, indicated with immunohistochemical and gene expression results. In both interval times, we also observed chondrogenesis and tissue formation after implanting unseeded bioengineered scaffolds, which denote that the presence of DC-ECM particles can even enhance attachment and migration of the host cells and induce chondrogenesis to them. To sum up, the incorporation of DC-ECM materials to tissue engineered constructs is a promising avenue to mimic the native tissue environment for regulation of cartilage regeneration in both in vivo and in vitro settings.
Subject(s)
Adult Stem Cells/cytology , Chondrogenesis/drug effects , Decellularized Extracellular Matrix/pharmacology , Tissue Scaffolds , Adipose Tissue/cytology , Adolescent , Adult , Animals , Animals, Outbred Strains , Cell Differentiation , Decellularized Extracellular Matrix/toxicity , Female , Foreign-Body Reaction/etiology , Humans , Male , Mice , Middle Aged , Nasal Cartilages , SOX9 Transcription Factor/biosynthesis , Subcutaneous Tissue , Tissue Scaffolds/adverse effects , Young AdultABSTRACT
Myocardial infarction (MI) is one of the leading causes of death worldwide. The complications associated with MI can lead to the formation of nonconductive fibrous scar tissues. Despite the great improvement in electroconductive biomaterials to increase the physiological function of bio-engineered cardiac tissues in vivo, there are still several challenges in creating a suitable scaffold with appropriate mechanical and electrical properties. In the current study, a highly hydrophilic fibrous scaffold composed of polycaprolactone/chitosan/polypyrrole (PCP) and combined with functionalized graphene, to provide superior conductivity and a stronger mechanical cardiopatch, is presented. The PCP/graphene (PCPG) patches were optimized to show mechanical and conductive properties close to the native myocardium. Also, the engineered patches showed strong capability as a drug delivery system. Heparin, an anticoagulant drug, was loaded within the fibrous patches, and the adsorption of the bovine serum albumin (BSA) protein was evaluated. The optimized cardiopatch shows great potential to be used to provide mechanical support and restore electromechanical coupling at the site of MI to maintain a normal cardiac function.
Subject(s)
Graphite , Animals , Biocompatible Materials , Cattle , Myocardium , Polymers , PyrrolesABSTRACT
Mechanical strength along with high biocompatibility and water absorbing are among main characteristics of a desirable scaffold for cartilage tissue engineering. Having these properties, polyvinyl alcohol (PVA) can be a good option for constructing cartilage tissue engineering scaffolds. In this study, PVA hydrogel was produced by freeze-thaw crosslinking method, and its mechanical properties such as viscoelastic and hyperplastic behavior, which cannot be obtained analytically, was investigated with a coupled finite element (FE)-optimization algorithm and stress relaxation experimental data. To obtain isotropic hyper-viscoelastic constitutive parameters of PVA scaffolds, the Mooney-Rivlin and Neo-Hooke strain energy functions, in which shear and bulk moduli varies with time, were applied. Results showed that predicted mechanical responses of scaffolds by the Mooney-Rivlin model better fitted stress-relaxation experiments than those obtained by Neo-Hooke one. Also, the properties obtained from the finite element model, such as the bulk and the shear moduli, showed that, after successful in vitro and in vivo experiments, PVA hydrogel may be introduced as a cartilage substitute for future tissue engineering therapies.
Subject(s)
Algorithms , Cartilage/cytology , Cartilage/drug effects , Finite Element Analysis , Polyvinyl Alcohol/pharmacology , Tissue Engineering/methods , Tissue Scaffolds , Polyvinyl Alcohol/chemistry , Tissue Scaffolds/chemistryABSTRACT
The mammalian retina contains multiple cellular layers, each carrying out a specific task. Such a controlled organization should be considered as a crucial factor for designing retinal therapies. The maintenance of retinal layered complexity through the use of scaffold-free techniques has recently emerged as a promising approach for clinical ocular tissue engineering. In an attempt to fabricate such layered retinal model, we are proposing herein a unique inkjet bioprinting system applied to the deposition of a photoreceptor cells (PRs) layer on top of a bioprinted retinal pigment epithelium (RPE), in a precise arrangement and without any carrier material. The results showed that, after bioprinting, both RPE and PRs were well positioned in a layered structure and expressed their structural markers, which was further demonstrated by ZO1, MITF, rhodopsin, opsin B, opsin R/G and PNA immunostaining, three days after bioprinting. We also showed that considerable amounts of human vascular endothelial growth factors (hVEGF) were released from the RPE printed layer, which confirmed the formation of a functional RPE monolayer after bioprinting. Microstructures of bioprinted cells as well as phagocytosis of photoreceptor outer segments by apical RPE microvilli were finally established through transmission electron microscopy (TEM) imaging. In summary, using this carrier-free bioprinting method, it was possible to develop a reasonable in vitro retina model for studying some sight-threatening diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).
Subject(s)
Bioprinting/methods , Retina/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Bioprinting/instrumentation , Cell Proliferation , Humans , Photoreceptor Cells/cytology , Photoreceptor Cells/metabolism , Printing, Three-Dimensional/instrumentation , Retina/metabolism , Retinal Pigment Epithelium/cytology , Rhodopsin/metabolism , Swine , Tissue Engineering/instrumentation , Vascular Endothelial Growth Factor A/metabolismABSTRACT
As a cellular-assembly technique, bioprinting has been extensively used in tissue engineering and regenerative medicine to construct hydrogel-based three-dimensional (3D) tissue-like models with prescribed geometry. Here, we introduced a unique direct-write bioprinting strategy to fabricate a bilayer flat tissue in a hydrogel-free approach. A printed retina pigmented epithelium layer (RPE) was applied as living biopaper for positioning a fibroblast layer without using any hydrogel in bioink. We adjusted the number of cells in the inkjet droplets in order to obtain a uniform printed cell layer and demonstrated the formation of a bilayer construct through confocal imaging. Since our printing system introduced low levels of shear stress to the cells, it did not have a negative effect on cell survival, although cell viability was generally lower than that of control group over 1 week post-printing. In conclusion, our novel direct-write bioprinting approach to spatiotemporally position different cellular layers may represent an efficient tool to develop living constructs especially for regeneration of complex flat tissues.
ABSTRACT
Current studies based on regenerative medicine suggest, decellularized extracellular matrix (DC-ECM) components can regulate cell phenotype. In this regard, it is believed, presence of cartilage extracellular matrix particles in culture condition could produce physical and biochemical supportive cues for chondrogenesis. In this study, DC-ECM nanoparticles with average size of 61.5± 22.4 nm were produced by decellularization and mechanical processing. Homogenous distribution and nanoscale size of yield particles were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) microscopy imaging. Chemical structure preservation of cartilage ECM after decellularization was also confirmed by typical Fourier transform infrared (FTIR) spectrum mapping. The influence these nanoparticles on chondrogenic response of chondrocyte cells was investigated by direct and indirect addition of nanoparticles to culture medium. A clinical devitalized cartilage powder (DV-ECM) was also used as a positive control. Totally, MTS results showed that direct and indirect presence of both DC-ECM and DV-ECM particles in culture medium enhanced cellular metabolic activity except on day one of culture. Furthermore, on day 21, SOX9 and COL2 expression of cultured chondrocytes in the medium containing DC-ECM nanoparticles were up-regulated in comparison to negative control, which was further confirmed by presence more frequent number of larger size lacunae in micromass spheroids. Our findings support the use of ECM nanoparticles as condition supplement in culture medium and injectable biomaterials, especially for cell-based therapies for cartilage regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2463-2471, 2018.
Subject(s)
Cartilage, Articular/metabolism , Chondrogenesis , Extracellular Matrix/metabolism , Nanoparticles/chemistry , Cartilage, Articular/ultrastructure , Cell Differentiation , Cell Shape , Cells, Cultured , Chondrocytes/cytology , Humans , Nanoparticles/ultrastructureABSTRACT
Biological and biomimetic decellularized scaffolds can mimic a natural tissue environment to derive cell proliferation and differentiation without eliciting adverse immune responses during tissue regeneration. Polymeric nanofibrous membranes also served as appropriate matrices for cellular behavior because of their resemblance to physical dimensions of natural extracellular matrix (ECM), while they often have insufficient biological cues to address the cellular phenotype. In this study, we designed bio-engineered membranes through covalent immobilization of decellularized ECM (DECM) particles on the surface of electrospun nanofibers and examined the ability of these composite materials for chondrogenesis. After successful chemical decellularization of human nasal septum cartilage constructs (hNSCs), mechanical processing was carried out and particles with a diameter mean size of 5.06 ± 2.70 µm were yielded. Poly hydroxyalkanoate (PHA) nanofibrous scaffolds were functionalized with DECM particles to mimic the natural motifs of cartilage ECM. Human adipose derived stem cells (hASCs) and human primary chondrocytes (hPChs) cultured on these biofunctional scaffolds showed a significant increase in collagen formation and chondrogenic marker expression after 21 days of cell culture. These results are exciting as they indicate the feasibility of creating bio-engineered scaffolds that may be non-immunogenic as a replacement tissue and have great potential for meeting new challenges in regenerative medicine, particularly in relation to cartilage reconstruction.
ABSTRACT
Interactions between Schwann cells (SCs) and scaffolds are important for tissue development during nerve regeneration, because SCs physiologically assist in directing the growth of regenerating axons. In this study, we prepared electrospun scaffolds combining poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) functionalized with either collagen I, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS), H-Tyr-Ile-Gly-Ser-Arg-NH2 (YIGSR), or H-Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile-OH (p20) neuromimetic peptides to mimic naturally occurring ECM motifs for nerve regeneration. Cells cultured on fibrous mats presenting these biomolecules showed a significant increase in metabolic activity and proliferation while exhibiting unidirectional orientation along the orientation of the fibers. Real-time PCR showed cells cultured on peptide-modified scaffolds had a significantly higher neurotrophin expression compared to those on untreated nanofibers. Our study suggests that biofunctionalized aligned PHB/PHBV nanofibrous scaffolds may elicit essential cues for SCs activity and could serve as a potential scaffold for nerve regeneration. From the clinical editor: Nanotechnology-based functionalized scaffolds represent one of the most promising approaches in peripheral nerve recovery, as well as spinal cord recovery. In this study, bio-functionalized and aligned PHB/PHBV nanofibrous scaffolds were found to elicit essential cues for Schwann cell activity, therefore could serve as a potential scaffold for nerve regeneration.
Subject(s)
Nanofibers , Peptides/chemistry , Polyhydroxyalkanoates/chemistry , Schwann Cells/cytology , Tissue Scaffolds , Enzyme-Linked Immunosorbent Assay , Humans , Microscopy, Electron, Scanning , Prohibitins , Spectroscopy, Fourier Transform InfraredABSTRACT
Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm(-1) can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.
Subject(s)
Biocompatible Materials , Nanofibers , Nerve Tissue , Tissue Engineering , Tissue Scaffolds , 3-Hydroxybutyric Acid/chemistry , Animals , Cell Proliferation , Gene Expression , Materials Testing , Nanofibers/chemistry , Nanofibers/ultrastructure , Prohibitins , Rats , Schwann Cells/cytology , Schwann Cells/metabolismABSTRACT
A critical element in tissue engineering involves the fabrication of a three-dimensional scaffold. The scaffold provides a space for new tissue formation, supports cellular ingrowth, and proliferation and mimics many roles of the extracellular matrix. Poly(3-hydroxybutyrate) (PHB) is the most thoroughly investigated member of the polyhydroxyalkanoates (PHAs) family that has various degrees of biocompatibility and biodegradability for tissue engineering applications. In this study, we fabricated PHB scaffolds by utilizing electrospinning and salt-leaching procedures. The behavior of monkey epithelial kidney cells (Vero) and mouse mesenchymal stem cells (mMSCs) on these scaffolds was compared by the MTS assay and scanning electron microscopy. Additionally, this study investigated the mechanical and physical properties of these scaffolds by measuring tensile strength and modulus, dynamic contact angle and porosity. According to our results, the salt-leached scaffolds showed more wettability and permeability, but inferior mechanical properties when compared with nanofibrous scaffolds. In terms of cell response, salt-leached scaffolds showed enhanced Vero cell proliferation, whereas both scaffolds responded similarly in the case of mMSCs proliferation. In brief, nanofibrous scaffolds can be a better substrate for cell attachment and morphology.