An injectable poly(DL-lactic-co-glycolic acid) (PLGA) system comprising both porous and protein-loaded microspheres capable of forming porous scaffolds at body temperature was developed for tissue regeneration purposes. Porous and non-porous (lysozyme loaded) PLGA microspheres were formulated to represent 'low molecular weight' 22-34 kDa, 'intermediate molecular weight' (IMW) 53 kDa and 'high molecular weight' 84-109 kDa PLGA microspheres. The respective average size of the microspheres was directly related to the polymer molecular weight. An initial burst release of lysozyme was observed from both microspheres and scaffolds on day 1. In the case of the lysozyme-loaded microspheres, this burst release was inversely related to the polymer molecular weight. Similarly, scaffolds loaded with 1 mg lysozyme/g of scaffold exhibited an inverse release relationship with polymer molecular weight. The burst release was highest amongst IMW scaffolds loaded with 2 and 3 mg/g. Sustained lysozyme release was observed after day 1 over 50 days (microspheres) and 30 days (scaffolds). The compressive strengths of the scaffolds were found to be inversely proportional to PLGA molecular weight at each lysozyme loading. Surface analysis indicated that some of the loaded lysozyme was distributed on the surfaces of the microspheres and thus responsible for the burst release observed. Overall the data demonstrates the potential of the scaffolds for use in tissue regeneration.
The development of patient-friendly alternatives to bone-graft procedures is the driving force for new frontiers in bone tissue engineering. Poly (dl-lactic-co-glycolic acid) (PLGA) and chitosan are well-studied and easy-to-process polymers from which scaffolds can be fabricated. In this study, a novel dual-application scaffold system was formulated from porous PLGA and protein-loaded PLGA/chitosan microspheres. Physicochemical and in vitro protein release attributes were established. The therapeutic relevance, cytocompatibility with primary human mesenchymal stem cells (hMSCs) and osteogenic properties were tested. There was a significant reduction in burst release from the composite PLGA/chitosan microspheres compared with PLGA alone. Scaffolds sintered from porous microspheres at 37 °C were significantly stronger than the PLGA control, with compressive strengths of 0.846 ± 0.272 MPa and 0.406 ± 0.265 MPa, respectively (p
Collagen type I is the most abundant matrix protein in the human body and is highly demanded in tissue engineering, regenerative medicine, and pharmaceutical applications. To meet the uprising demand in biomedical applications, collagen type I has been isolated from mammalians (bovine, porcine, goat and rat) and non-mammalians (fish, amphibian, and sea plant) source using various extraction techniques. Recent advancement enables fabrication of collagen scaffolds in multiple forms such as film, sponge, and hydrogel, with or without other biomaterials. The scaffolds are extensively used to develop tissue substitutes in regenerating or repairing diseased or damaged tissues. The 3D scaffolds are also used to develop in vitro model and as a vehicle for delivering drugs or active compounds.
Studies of peri-implant soft tissue on in vivo models are commonly based on histological sections prepared using undecalcified or 'fracture' techniques. These techniques require the cutting or removal of implant during the specimen preparation process. The aim of this study is to explore a new impression technique that does not require any cutting or removal of implant for contour analysis of soft tissue around four types of titanium (Ti) surface roughness using an in vitro three-dimensional oral mucosal model (3D OMM).
Most biological tissues grow by the synthesis of new material close to the tissue's interface, where spatial interactions can exert strong geometric influences on the local rate of growth. These geometric influences may be mechanistic or cell behavioural in nature. The control of geometry on tissue growth has been evidenced in many in vivo and in vitro experiments, including bone remodelling, wound healing, and tissue engineering scaffolds. In this paper, we propose a generalisation of a mathematical model that captures the mechanistic influence of curvature on the joint evolution of cell density and tissue shape during tissue growth. This generalisation allows us to simulate abrupt topological changes such as tissue fragmentation and tissue fusion, as well as three dimensional cases, through a level-set-based method. The level-set method developed introduces another Eulerian field than the level-set function. This additional field represents the surface density of tissue-synthesising cells, anticipated at future locations of the interface. Numerical tests performed with this level-set-based method show that numerical conservation of cells is a good indicator of simulation accuracy, particularly when cusps develop in the tissue's interface. We apply this new model to several situations of curvature-controlled tissue evolutions that include fragmentation and fusion.
Guided bone regeneration (GBR) scaffolds are futile in many clinical applications due to infection problems. In this work, we fabricated GBR with an anti-infective scaffold by ornamenting 2D single crystalline bismuth-doped nanohydroxyapatite (Bi-nHA) rods onto segmented polyurethane (SPU). Bi-nHA with high aspect ratio was prepared without any templates. Subsequently, it was introduced into an unprecedented synthesized SPU matrix based on dual soft segments (PCL-b-PDMS) of poly(ε-caprolactone) (PCL) and poly(dimethylsiloxane) (PDMS), by an in situ technique followed by electrospinning to fabricate scaffolds. For comparison, undoped pristine nHA rods were also ornamented into it. The enzymatic ring-opening polymerization technique was adapted to synthesize soft segments of PCL-b-PDMS copolymers of SPU. Structure elucidation of the synthesized polymers is done by nuclear magnetic resonance spectroscopy. Sparingly, Bi-nHA ornamented scaffolds exhibit tremendous improvement (155%) in the mechanical properties with excellent antimicrobial activity against various human pathogens. After confirmation of high osteoconductivity, improved biodegradation, and excellent biocompatibility against osteoblast cells (in vitro), the scaffolds were implanted in rabbits by subcutaneous and intraosseous (tibial) sites. Various histological sections reveal the signatures of early cartilage formation, endochondral ossification, and rapid bone healing at 4 weeks of the critical defects filled with ornamented scaffold compared to SPU scaffold. This implies osteogenic potential and ability to provide an adequate biomimetic microenvironment for mineralization for GBR of the scaffolds. Organ toxicity studies further confirm that no tissue architecture abnormalities were observed in hepatic, cardiac, and renal tissue sections. This finding manifests the feasibility of fabricating a mechanically adequate nanofibrous SPU scaffold by a biomimetic strategy and the advantages of Bi-nHA ornamentation in promoting osteoblast phenotype progression with microbial protection (on-demand) for GBR applications.
Three-dimensional assembly of graphene hydrogel is rapidly attracting the interest of researchers because of its wide range of applications in energy storage, electronics, electrochemistry, and waste water treatment. Information on the use of graphene hydrogel for biological purposes is lacking, so we conducted a preliminary study to determine the suitability of graphene hydrogel as a substrate for cell growth, which could potentially be used as building blocks for biomolecules and tissue engineering applications.
The in vivo biocompatibility and toxicity of PVA/NOCC scaffold were tested by comparing them with those of a biocompatible inert material HAM in a rat model. On Day 5, changes in the blood parameters of the PVA/NOCC-implanted rats were significantly higher than those of the control. The levels of potassium, creatinine, total protein, A/G, hemoglobulin, erythrocytes, WBC, and platelets were not significantly altered in the HAM-implanted rats, when compared with those in the control. On Day 10, an increase in potassium, urea, and GGT levels and a decrease in ALP, platelet, and eosinophil levels were noted in the PVA/NOCC-implanted rats, when compared with control. These changes were almost similar to those noted in the HAM-implanted rats, except for the unaltered potassium and increased neutrophil levels. On Day 15, the total protein, A/G, lymphocyte, monocyte, and eosinophil levels remained unaltered in the PVA/NOCC-implanted rats, whereas urea, A/G, WBC, lymphocyte, and monocyte levels remained unchanged in the HAM-implanted rats. Histology and immunohistochemistry analyses revealed inflammatory infiltration in the PVA/NOCC-implanted rats, but not in the HAM-implanted rats. Although a low toxic tissue response was observed in the PVA/NOCC-implanted rats, further studies are necessary to justify the use of this material in tissue engineering applications.
Electrospinning is a promising technique for the fabrication of bioscaffolds in tissue engineering applications. Pertaining issues of multiple polymer jets and bending instabilities result in random paths which lend poor controllability over scaffolds morphology for affecting the porosity and mechanical stability. The present study alleviates these challenges by demonstrating a novel self-directing single jet taking a specifically patterned path to deposit fibers into circular and uniform scaffolds without tuning any externally controlled parameters. High-speed camera observation revealed that the charge retention and dissipation on the collected fibers caused rapid autojet switching between the two jetting modes, namely, a microcantilever-like armed jet motion and a whipping motion, which sequentially expand the area and thickness of the scaffolds, respectively, in a layered-like fashion. The physical properties showed that the self-switching dual-jet modes generated multilayered microfibrous scaffolds (MFSs) with dual morphologies and varied fiber packing density, thereby establishing the gradient porosity and mechanical strength (through buckled fibers) in the scaffolds. In vitro studies showed that as-spun scaffolds are cell-permeable hierarchical 3D microporous structures enabling lateral cell seeding into multiple layers. The cell proliferation on days 6 and 9 increased 21% and 38% correspondingly on MFSs than on nanofibrous scaffolds (NFSs) done by conventional multijets electrospinning. Remarkably, this novel and single-step process is highly reproducible and tunable for developing fibrous scaffolds for tissue engineering applications.
Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries) Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin film with collagen fibrils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achilles tendon using 0.35M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further analysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDS) confirms the presence of triple helix structure of col I, similar to commercially available rat tail col I. Drying the OTC solution at 37°C resulted in formation of a thin film with randomly orientated collagen fibrils (random collagen film; RCF). Introduction of unidirectional mechanical intervention using a platform rocker prior to drying facilitated the fabrication of a film with aligned orientation of collagen fibril (aligned collagen film; ACF). It was shown that both RCF and ACF significantly enhanced human dermal fibroblast (HDF) attachment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative source of col I to fabricate scaffold for tissue engineering applications.
Fabrication of composite scaffolds is one of the strategies proposed to enhance the functionality of tissue-engineered scaffolds for improved tissue regeneration. By combining multiple elements together, unique biomimetic scaffolds with desirable physical and mechanical properties can be tailored for tissue-specific applications. Despite having a highly porous structure, the utility of electrospun fibers (EF) as scaffold is usually hampered by their insufficient mechanical strength. In this study, we attempted to produce a mechanically competent scaffold with cell-guiding ability by fabricating aligned poly lactic-co-glycolic acid (PLGA) fibers on decellularized human amniotic membrane (HAM), known to possess favorable tensile and wound healing properties. Decellularization of HAM in 18.75 μg/mL of thermolysin followed by a brief treatment in 0.25 M sodium hydroxide efficiently removed the amniotic epithelium and preserved the ultrastructure of the underlying extracellular matrix. The electrospinning of 20% (w/v) PLGA 50:50 polymer on HAM yielded beadless fibers with straight morphology. Subsequent physical characterization revealed that EF-HAM scaffold with a 3-min fabrication had the most aligned fibers with the lowest fiber diameter in comparison with EF-HAM 5- and 7-min scaffolds. Hydrated EF-HAM scaffolds with 3-min deposition had a greater tensile strength than the other scaffolds despite having thinner fibers. Nevertheless, wet HAM and EF-HAMs regardless of the fiber thicknesses had a significantly lower Young's modulus, and hence, a higher elasticity compared with dry HAM and EF-HAMs. Biocompatibility analysis showed that the viability and migration rate of skeletal muscle cells on EF-HAMs were similar to control and HAM alone. Skeletal muscle cells seeded on HAM were shown to display random orientation, whereas cells on EF-HAM scaffolds were oriented along the alignment of the electrospun PLGA fibers. In summary, besides having good mechanical strength and elasticity, EF-HAM scaffold design decorated with aligned fiber topography holds a promising potential for use in the development of aligned tissue constructs.
The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.
Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h-1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h-1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.
The osteogenic potential of human adipose-derived stem cells (HADSCs) co-cultured with human osteoblasts (HOBs) using selected HADSCs/HOBs ratios of 1:1, 2:1, and 1:2, respectively, is evaluated. The HADSCs/HOBs were seeded on electrospun three-dimensional poly[(R)-3-hydroxybutyric acid] (PHB) blended with bovine-derived hydroxyapatite (BHA). Monocultures of HADSCs and HOBs were used as control groups. The effects of PHB-BHA scaffold on cell proliferation and cell morphology were assessed by AlamarBlue assay and field emission scanning electron microscopy. Cell differentiation, cell mineralization, and osteogenic-related gene expression of co-culture HADSCs/HOBs were examined by alkaline phosphatase (ALP) assay, alizarin Red S assay, and quantitative real time PCR, respectively. The results showed that co-culture of HADSCs/HOBs, 1:1 grown into PHB-BHA promoted better cell adhesion, displayed a significant higher cell proliferation, higher production of ALP, extracellular mineralization and osteogenic-related gene expression of run-related transcription factor, bone sialoprotein, osteopontin, and osteocalcin compared to other co-culture groups. This result also suggests that the use of electrospun PHB-BHA in a co-culture HADSCs/HOBs system may serve as promising approach to facilitate osteogenic differentiation activity of HADSCs through direct cell-to-cell contact with HOBs.
In this study, antibacterial characteristic of silver/poly (lactic acid) nanocomposite (Ag/PLA-NC) films was investigated, while silver nanoparticles (Ag-NPs) were synthesized into biodegradable PLA via chemical reduction method in diphase solvent. Silver nitrate and sodium borohydride were respectively used as a silver precursor and reducing agent in the PLA, which acted as a polymeric matrix and stabilizer. Meanwhile, the properties of Ag/PLA-NCs were studied as a function of the Ag-NP weight percentages (8, 16, and 32 wt% respectively), in relation to the use of PLA. The morphology of the Ag/PLA-NC films and the distribution of the Ag-NPs were also characterized. The silver ions released from the Ag/PLA-NC films and their antibacterial activities were scrutinized. The antibacterial activities of the Ag/PLA-NC films were examined against Gram-negative bacteria (Escherichia coli and Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) by diffusion method using Muller-Hinton agar. The results indicated that Ag/PLA-NC films possessed a strong antibacterial activity with the increase in the percentage of Ag-NPs in the PLA. Thus, Ag/PLA-NC films can be used as an antibacterial scaffold for tissue engineering and medical application.
Understanding the bioelectrical properties of bone tissue is key to developing new treatment strategies for bone diseases and injuries, as well as improving the design and fabrication of scaffold implants for bone tissue engineering. The bioelectrical properties of bone tissue can be attributed to the interaction of its various cell lineages (osteocyte, osteoblast and osteoclast) with the surrounding extracellular matrix, in the presence of various biomechanical stimuli arising from routine physical activities; and is best described as a combination and overlap of dielectric, piezoelectric, pyroelectric and ferroelectric properties, together with streaming potential and electro-osmosis. There is close interdependence and interaction of the various electroactive and electrosensitive components of bone tissue, including cell membrane potential, voltage-gated ion channels, intracellular signaling pathways, and cell surface receptors, together with various matrix components such as collagen, hydroxyapatite, proteoglycans and glycosaminoglycans. It is the remarkably complex web of interactive cross-talk between the organic and non-organic components of bone that define its electrophysiological properties, which in turn exerts a profound influence on its metabolism, homeostasis and regeneration in health and disease. This has spurred increasing interest in application of electroactive scaffolds in bone tissue engineering, to recapitulate the natural electrophysiological microenvironment of healthy bone tissue to facilitate bone defect repair.
Culture scaffolds allow microalgae cultivation with minimum water requirement using the air-liquid interface approach. However, the stability of cellulose-based scaffolds in microalgae cultivation remains questionable. In this study, the stability of regenerated cellulose culture scaffolds was enhanced by adjusting TiO2 loading and casting gap. The membrane scaffolds were synthesized using cellulose dissolved in NaOH/urea aqueous solution with various loading of TiO2 nanoparticles. The TiO2 nanoparticles were embedded into the porous membrane scaffolds as proven by Fourier transform infrared spectra, scanning electron microscopic images, and energy-dispersive X-ray spectra. Although surface hydrophilicity and porosity were enhanced by increasing TiO2 and casting gap, the scaffold pore size was reduced. Cellulose membrane scaffold with 0.05 wt% of TiO2 concentration and thickness of 100 μm attained the highest percentage of Navicula incerta growth rate, up to 37.4%. The membrane scaffolds remained stable in terms of weight, porosity and pore size even they were immersed in acidic solution, hydrogen peroxide or autoclaved at 121 °C for 15 min. The optimal cellulose membrane scaffold is with TiO2 loading of 0.5 wt% and thickness of 100 μm, resulting in supporting the highest N. incerta growth rate and and exhibits good membrane stability.
Given their highly porous nature and excellent water retention, hydrogel-based biomaterials can mimic critical properties of the native cellular environment. However, their potential to emulate the electromechanical milieu of native tissues or conform well with the curved topology of human organs needs to be further explored to address a broad range of physiological demands of the body. In this regard, the incorporation of nanomaterials within hydrogels has shown great promise, as a simple one-step approach, to generate multifunctional scaffolds with previously unattainable biological, mechanical, and electrical properties. Here, recent advances in the fabrication and application of nanocomposite hydrogels in tissue engineering applications are described, with specific attention toward skeletal and electroactive tissues, such as cardiac, nerve, bone, cartilage, and skeletal muscle. Additionally, some potential uses of nanoreinforced hydrogels within the emerging disciplines of cyborganics, bionics, and soft biorobotics are highlighted.
One of the novel and progressive technology employed in pharmaceutical manufacturing, design of medical device and tissue engineering is three-dimensional (3D) printing. 3D printing technologies provide great advantages in 3D scaffolds fabrication over traditional methods in the control of pore size, porosity, and interconnectivity. Various techniques of 3D-printing include powder bed fusion, fused deposition modeling, binder deposition, inkjet printing, photopolymerization and many others which are still evolving. 3D-printing technique been employed in developing immediate release products, various systems to deliver multiple release modalities etc. 3D printing has opened the door for new generation of customized drug delivery with built-in flexibility for safer and effective therapy. Our mini-review provides a quick snapshot on an overview of 3D printing, various techniques employed, applications and its advancements in pharmaceutical sciences.