Successful Tissue Engineering Research Articles

Differential response of human blood leukocytes to brushite, monetite, and calcium polyphosphate biomaterials.

Calcium phosphate-based biomaterials are extensively used for bone replacement and regeneration in orthopedic, dental, and maxillofacial surgical applications. The injury induced by surgical implantation of bone replacement graft materials initiates a cascade of host responses, starting with blood-biomaterial contact, protein adsorption on the material surface, blood coagulation, and leukocyte responses. During the initial acute inflammatory response, polymorphonuclear neutrophils (PMNs) and monocytes, abundant circulating leukocytes of the myeloid lineage, are recruited to the site of inflammation. In addition to responding to pathogenic challenges, these cells respond to particulate substances within the body including crystals of monosodium urate (MSU). Host responses toward grafts impact short- and long-term success in tissue engineering and regenerative applications. Although multinucleated osteoclasts, formed by monocyte/macrophage fusion, are generally thought to be responsible for resorption of implant biomaterials, the ability of different biomaterials to trigger PMNs, which are invariably present at the early stages after implant surgery, and are abundant in the oral cavity, has never been tested. In this article, we present analysis of the response of human blood-derived PMNs and monocytes toward brushite, monetite, and calcium polyphosphate (CPP) biomaterial substrates and compare this to the response to MSU crystals, the latter serving as a positive control. Employing multicolor flow cytometry to look at PMN and monocyte cell surface markers of activation to gauge the response to different biomaterials, we found that both types of myeloid cells are highly activated after exposure to brushite, monetite, and MSU but not CPP. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:253-262, 2020.

Mimicking the Annulus Fibrosus Using Electrospun Polyester Blended Scaffolds.

Treatments to alleviate chronic lower back pain, caused by intervertebral disc herniation as a consequence of degenerate annulus fibrosus (AF) tissue, fail to provide long-term relief and do not restore tissue structure or function. This study aims to mimic the architecture and mechanical environment of AF tissue using electrospun fiber scaffolds made from synthetic biopolymers-poly(ε-caprolactone) (PCL) and poly(L-lactic) acid (PLLA). Pure polymer and their blends (PCL%:PLLA%; 80:20, 50:50, and 20:80) are studied and material properties-fiber diameter, alignment, % crystallinity, tensile strength, and water contact angle-characterized. Tensile properties of fibers angled at 0°, 30°, and 60° (single layer scaffolds), and ±0°, ±30°, and ±60° (bilayer scaffolds) yield significant differences, with PCL being significantly stiffer with the addition of PLLA, and bilayer scaffolds considerably stronger. Findings suggest PCL:PLLA 50:50 fibers are similar to human AF properties. Furthermore, in vitro culture of AF cells on 50:50 fibers demonstrates attachment and proliferation over seven days. The optimal polymer composition for production of scaffolds that closely mimic AF tissue both structurally, mechanically, and which also support and guide favorable cell phenotype is identified. This study takes a step closer towards successful AF tissue engineering and a long-term treatment for sufferers of chronic back pain.

Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers

Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.

Behavior of Mesenchymal Stem Cells Obtained From Dental Tissues: A Review of the Literature

The success of tissue engineering in combination with tissue regeneration depends on the behavior and cellular activity in the biological processes developed within a structure that functions as a support, better known as scaffolds, or directly at the site of the injury. The cell-cell and cell-biomaterial interaction are key factors for the induction of a specific cell behavior, together with the bioactive factors that allow the formation of the desired tissue. Mesenchymal Stem Cells (MSC) can be isolated from the umbilical cord and bone marrow; however, the behavior of Dental Pulp Stem Cells (DPSC) has been shown to have a high potential for the formation of bone tissue, and these cells have even been able to induce the process of angiogenesis. Advances in periodontal regeneration, dentin-pulp complex, and craniofacial bone defects through the induction of MSC obtained from tooth structures in in vitro-in vivo studies have permitted the obtaining of clinical evidence of the achievements obtained to date.

Tissue Engineering for the Ovine Rotator Cuff: Surgical Anatomy, Approach, Implantation and Histology Technique, along with Review of Literature

Aim: Tissue engineering is a contemporary topic, which is widely discussed in shoulder surgery. The preclinical success of tissue engineering has not yet fully translated to clinical studies. In this study, we present our experience in this subject and discuss measurements to analyze the sheep tissue as comparative model. We also present a comprehensive systematic review of the literature in relation to tissue engineering and rotator cuff. Method: We studied the anatomy, histology and surgical approach of 24 infraspinatus tendons specimens in sheep and focused on certain measurements such as: (1) size of the normal tendon; (2) diameter of the normal footprint; and (3) bone hardness of the footprint using a durometer. Blood from six sheep was obtained and processed to produce platelet rich plasma using both the Harvest Smartprep and Cascade kit. We then outlined an approach to the infraspinatus tendon footprint in preparation for implantation of a biological augmentation material, which was repaired using a double row technique. This was followed by a histological analysis. Results: The average measurements of the footprint were 21 mm ×21 mm, the tendon length was 35.1 mm and the width proximal and distal was 21.9 and 14 mm, respectively. The average bone hardness at the 12, 3, 6, and 9 o’clock position was 64.1, 52.3, 50.1, and 37.7 respectively. Central footprint and lateral edge hardness was 44.8 and 43.4. Only the Harvest Smartprep and using a modified method, was able to produce a platelet concentration factor of 4. The Cascade method was unable to concentrate sheep blood. Conclusion: The sheep model is a suitable tissue for studying the rotator cuff. The researcher must be aware of certain interspecies caveats. Clinical tissue engineering and surgical techniques must be modified in order to be suitable when using sheep model.

Biocompatibility of alumina-based biomaterials-A review.

In today's medicine world, alumina-based biomaterials owing to their excellent biomechanical, and biocompatibility properties play a key role in biomedical engineering. However, the literature still suffers from not having a valid database regarding the protein adsorption and subsequently cell responses to these surfaces. Proteins by adsorption on biomaterials surfaces start interpreting the construction and also arranging the biomaterials surfaces into a biological language. Hence, the main concentration of this review is on the protein adsorption and subsequently cell responses to alumina's surface, which has a wide range biomedical applications, especially in dentistry and orthopedic applications. In the presented review article, the general principles of foreign body response mechanisms, and also the role of adsorbed proteins as key players in starting interactions between cells and alumina-based biomaterials will be discussed in detail. In addition, the essential physicochemical, and mechanical properties of alumina surfaces which significantly impact on proteins and cells responses as well as the recent studies that have focused on the biocompatibility of alumina will be given. An in depth understanding of how the immune system interacts with the surface of alumina could prove the pivotal importance of the biocompatibility of alumina on its success in tissue engineering after implantation in body.

Lattice and continuum modelling of a bioactive porous tissue scaffold.

A contemporary procedure to grow artificial tissue is to seed cells onto a porous biomaterial scaffold and culture it within a perfusion bioreactor to facilitate the transport of nutrients to growing cells. Typical models of cell growth for tissue engineering applications make use of spatially homogeneous or spatially continuous equations to model cell growth, flow of culture medium, nutrient transport and their interactions. The network structure of the physical porous scaffold is often incorporated through parameters in these models, either phenomenologically or through techniques like mathematical homogenization. We derive a model on a square grid lattice to demonstrate the importance of explicitly modelling the network structure of the porous scaffold and compare results from this model with those from a modified continuum model from the literature. We capture two-way coupling between cell growth and fluid flow by allowing cells to block pores, and by allowing the shear stress of the fluid to affect cell growth and death. We explore a range of parameters for both models and demonstrate quantitative and qualitative differences between predictions from each of these approaches, including spatial pattern formation and local oscillations in cell density present only in the lattice model. These differences suggest that for some parameter regimes, corresponding to specific cell types and scaffold geometries, the lattice model gives qualitatively different model predictions than typical continuum models. Our results inform model selection for bioactive porous tissue scaffolds, aiding in the development of successful tissue engineering experiments and eventually clinically successful technologies.

Cyclic Uniaxial Tensile Strain Enhances the Mechanical Properties of Engineered, Scaffold-Free Tendon Fibers.

The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain reaction to measure the expression of tendon-specific makers, scleraxis and tenomodulin. Fiber peak stress, elastic modulus, toughness, and nuclear aspect ratio increased with the presence and duration of loading, while failure strain, toe-in strain, and nuclear area were unchanged. These biomechanical results suggest that cyclic strain promotes matrix deposition in a manner that increases the fiber resistance to stretch, but preserves fiber extensibility over the 7-day loading period. Over 7 days of loading, the scleraxis and tenomodulin expression increased drastically. Histologically, while there was no immediate difference in nuclear area with the addition of loading, nuclear aspect ratio significantly increased with loading duration, such that nuclei became progressively more elongated to the long axis of the fiber. Together with our biomechanical findings, such nuclear deformation suggests that cyclic strain elicits a mechanotransductive response, particularly one that modulates gene expression to promote matrix deposition during fiber development.

Bioinspired liver scaffold design criteria

ABSTRACTMaintaining hepatic functional characteristics in-vitro is considered one of the main challenges in engineering liver tissue. As hepatocytes cultured ex-vivo are deprived of their native extracellular matrix (ECM) milieu, developing scaffolds that mimic the biomechanical and physicochemical properties of the native ECM is thought to be a promising approach for successful tissue engineering and regenerative medicine applications. On the basis that the decellularized liver matrix represents the ideal design template for engineering bioinspired hepatic scaffolds, to derive quantitative descriptors of liver ECM architecture, we characterised decellularised liver matrices in terms of their biochemical, viscoelastic and structural features along with porosity, permeability and wettability. Together, these data provide a unique set of quantitative design criteria which can be used to generate guidelines for fabricating biomaterial scaffolds for liver tissue engineering. As proof-of-concept, we investigated hepatic cell response to substrate viscoelasticity. On collagen hydrogels mimicking decellularised liver mechanics, cells showed superior morphology, higher viability and albumin secretion than on stiffer and less viscous substrates. Although scaffold properties are generally inspired by those of native tissues, our results indicate significant differences between the mechano-structural characteristics of untreated and decellularised hepatic tissue. Therefore, we suggest that design rules - such as mechanical properties and swelling behaviour - for engineering biomimetic scaffolds be re-examined through further studies on substrates matching the features of decellularized liver matrices.

Thermoresponsive Citrate-Based Graphene Oxide Scaffold Enhances Bone Regeneration from BMP9-Stimulated Adipose-Derived Mesenchymal Stem Cells.

Effective bone tissue engineering is important to overcome the unmet clinical challenges as more than 1.6 million bone grafts are done annually in the United States. Successful bone tissue engineering needs minimally three critical constituents: osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds. Osteogenic progenitors are derived from multipotent mesenchymal stem cells (MSCs), which can be prepared from numerous tissue sources, including adipose tissue. We previously showed that BMP9 is the most osteogenic BMP and induces robust bone formation of immortalized mouse adipose-derived MSCs entrapped in a citrate-based thermoresponsive hydrogel referred to as PPCNg. As graphene and its derivatives emerge as promising biomaterials, here we develop a novel thermosensitive and injectable hybrid material by combining graphene oxide (GO) with PPCNg (designated as GO-P) and characterize its ability to promote bone formation. We demonstrate that the thermoresponsive behavior of the hybrid material is maintained while effectively supporting MSC survival and proliferation. Furthermore, GO-P induces early bone-forming marker alkaline phosphatase (ALP) and potentiates BMP9-induced expression of osteogenic regulators and bone markers as well as angiogenic factor VEGF in MSCs. In vivo studies show BMP9-transduced MSCs entrapped in the GO-P scaffold form well-mineralized and highly vascularized trabecular bone. Thus, these results indicate that GO-P hybrid material may function as a new biocompatible, injectable scaffold with osteoinductive and osteoconductive activities for bone regeneration.

Biochemical and biomechanical characterisation of equine cervical facet joint cartilage.

The equine cervical facet joint is a site of significant pathology. Located bilaterally on the dorsal spine, these diarthrodial joints work in conjunction with the intervertebral disc to facilitate appropriate spinal motion. Despite the high prevalence of pathology in this joint, the facet joint is understudied and thus lacking in viable treatment options. The goal of this study was to characterise equine facet joint cartilage and provide a comprehensive database describing the morphological, histological, biochemical and biomechanical properties of this tissue. Descriptive cadaver studies. A total of 132 facet joint surfaces were harvested from the cervical spines of six skeletally mature horses (11 surfaces per animal) for compiling biomechanical and biochemical properties of hyaline cartilage of the equine cervical facet joints. Gross morphometric measurements and histological staining were performed on facet joint cartilage. Creep indentation and uniaxial strain-to-failure testing were used to determine the biomechanical compressive and tensile properties. Biochemical assays included quantification of total collagen, sulfated glycosaminoglycan and DNA content. The facet joint surfaces were ovoid in shape with a flat articular surface. Histological analyses highlighted structures akin to articular cartilage of other synovial joints. In general, biomechanical and biochemical properties did not differ significantly between the inferior and superior joint surfaces as well as among spinal levels. Interestingly, compressive and tensile properties of cervical facet articular cartilage were lower than those of articular cartilage from other previously characterised equine joints. Removal of the superficial zone reduced the tissue's tensile strength, suggesting that this zone is important for the tensile integrity of the tissue. Facet surfaces were sampled at a single, central location and do not capture the potential topographic variation in cartilage properties. This is the first study to report the properties of equine cervical facet joint cartilage and may serve as the foundation for the development of future tissue-engineered replacements as well as other treatment strategies.

Acrylic acid grafted tamarind kernel polysaccharide-based hydrogel for bone tissue engineering in absence of any osteo-inducing factors

ABSTRACTPurpose: With increased life expectancy, disorders in lifestyle and other clinical conditions, and the changes in the connective tissues such as in bone, impose diverse biomedical problems. Cells belong to osteogenic lineages are extremely specific for their surface requirements. Therefore, suitable surfaces are the critical bottle neck for successful bone tissue engineering. This study involves assessment of polysaccharide-based hydrogel which effectively allows growth, differentiation and mineralisation of osteogenic cells even in the absence of osteogenic inducing factors. Materials and methods: Tamarind Kernel Polysaccharide was grafted with acrylic acid at different mole ratio. The critical parameter, surface morphology for bio application was assessed by SEM. MTT assay has been performed with hydrogels on Saos-2 cells. The biocompatibility and adhesion of different cell lines (F-11, Saos-2, Raw 264.7 and MSCs) on hydrogel surface was performed by Phalloidin and DAPI staining. Further the differentiation, mineralization and expression of different osteogenic markers, ALP assay, Alizarin Red staining and q-PCR was performed. Results: The hydrogels show highly porous and interconnected pores. MTT assay demonstrates the hydrogel have no cytotoxicity towards Saos-2 cells and are suitable for proliferation of different lineage of cell lines. ALP, Alizarin red staining and q-PCR assay shows that the hydrogel surface enhances the differentiation, mineralization and expression of different osteogenic genes in Saos-2 cells in the absence of any osteogenic inducing factors. Conclusion Synthesized hydrogel surface triggers signalling events towards osteogenesis even in the absence of added growth factors. We proposed that this material can be used for effective bone tissue engineering in vitro at low cost.

Amyloid Fibrils: Versatile Biomaterials for Cell Adhesion and Tissue Engineering Applications.

Extracellular matrices (ECM) play an enormous role in any living system, controlling various factors and eventually fates of cells. ECM regulates cell fate by providing constant exogenous signals altering intracellular signal transduction for diverse pathways including proliferation, migration, differentiation, and apoptosis. Biomaterial scaffolds are designed to mimic the natural extracellular matrix such that the cells could recapitulate natural events alike their natural niche. Therefore, the success of tissue engineering is largely dependent on how one can engineer the natural matrix properties at nanoscale precision. In this aspect, several recent studies have suggested that, as long as amyloid fibrils are not toxic, they can be utilized for cell adhesion and tissue engineering applications due to its ECM mimetic surface topography and ability to mediate active cell adhesion via focal adhesions. Although historically associated with human diseases, amyloids have presently emerged as one of the excellent biomaterials evolved in nature. In this review, we focus on the recent advances of amyloid-based biomaterials for cell adhesion and tissue engineering applications.

MP61-03 A BIOFUNCTIONAL VASCULAR SCAFFOLD FOR REPLACING SMALL-DIAMETER BLOOD VESSELS

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation & Vascular Surgery I1 Apr 2018MP61-03 A BIOFUNCTIONAL VASCULAR SCAFFOLD FOR REPLACING SMALL-DIAMETER BLOOD VESSELS Young Min Ju, Ickhee Kim, John Jackson, Anthony Atala, James Yoo, and Sang Jin Lee Young Min JuYoung Min Ju More articles by this author , Ickhee KimIckhee Kim More articles by this author , John JacksonJohn Jackson More articles by this author , Anthony AtalaAnthony Atala More articles by this author , James YooJames Yoo More articles by this author , and Sang Jin LeeSang Jin Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1982AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Disease of the renal vascular system has resulted in a clear clinical need for the development of functional vascular substitutes. We have previously fabricated a vascular scaffold by electrospinning of poly(e-caprolactone) (PCL) combined with collagen. In this study, we hypothesized that multiple bioconjugation of endothelial progenitor cells (EPC)/endothelial cell (EC) specific antibodies and anti-thrombogenic agents onto the vascular scaffold could facilitate in situ endothelialization while preventing blood clotting. METHODS Vascular scaffolds were fabricated by electrospinning of poly(e-caprolactone) (PCL) combined with collagen. Bioconjugation of endothelial progenitor cells (EPC)/endothelial cell (EC) specific antibodies and anti-thrombogenic agents were performed onto the vascular scaffold. The bioconjugated scaffolds were implanted in a sheep model of vascular defect. RESULTS The biofunctionalized scaffolds conjugated with EPC/EC-specific antibodies and heparin were able to achieve effective EPC/EC capturing and anti-thrombogenesis. In vivo experiment showed that these vascular scaffolds maintained a high degree of patency and structural integrity without eliciting a histologic inflammatory response over the course of 6-month period in sheep. Moreover, the matured EC coverage on the lumen and smooth muscle layer were observed at 6 months after implantation. CONCLUSIONS We demonstrate that the generation of the biofunctional vascular scaffold, along with existing tools for selection and directed immobilization of EPC/EC-specific antibodies and anti-thrombogenic agent, could provide the necessary components for successful tissue engineering of small diameter blood vessels in the renal vascular system. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e824 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Young Min Ju More articles by this author Ickhee Kim More articles by this author John Jackson More articles by this author Anthony Atala More articles by this author James Yoo More articles by this author Sang Jin Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

In vitro evaluation of a bone morphogenetic protein‑2 nanometer hydroxyapatite collagen scaffold for bone regeneration.

Scaffold fabrication and biocompatibility are crucial for successful bone tissue engineering. Nanometer hydroxyapatite (nHAP) combined with collagen (COL) is frequently utilized as a suitable osseous scaffold material. Furthermore, growth factors, including bone morphogenetic protein-2 (BMP-2), are used to enhance the scaffold properties. The present study used blending and freeze-drying methods to develop a BMP-2-nHAP-COL scaffold. An ELISA was performed to determine the BMP-2 release rate from the scaffold. Flow cytometry was used to identify rat bone marrow-derived mesenchymal stem cells (BMSCs) prior to their combination with the scaffold. Scanning electron microscopy was used to observe the scaffold structure and BMSC morphology following seeding onto the scaffold. BMSCs were also used to assess the biological compatibility of the scaffold in vitro. BMP-2-nHAP-COL and nHAP-COL scaffolds were assessed alongside the appropriate control groups. Cells were counted to determine early cell adhesion. Cell Counting kit-8 and alkaline phosphatase assays were used to detect cell proliferation and differentiation, respectively. Gross morphology confirmed that the BMP-2-nHAP-COL scaffold microstructure conformed to the optimal characteristics of a bone tissue engineering scaffold. Furthermore, the BMP-2-nHAP-COL scaffold exhibited no biological toxicity and was demonstrated to promote BMSC adhesion, proliferation and differentiation. The BMP-2-nHAP-COL scaffold had good biocompatibility in vitro, and may therefore be modified further to construct an optimized scaffold for future bone tissue engineering.

Synthetic design of growth factor sequestering extracellular matrix mimetic hydrogel for promoting in vivo bone formation

Synthetic scaffolds that possess an intrinsic capability to protect and sequester sensitive growth factors is a primary requisite for developing successful tissue engineering strategies. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) is highly susceptible to premature degradation and to provide a meaningful clinical outcome require high doses that can cause serious side effects. We discovered a unique strategy to stabilize and sequester rhBMP-2 by enhancing its molecular interactions with hyaluronic acid (HA), an extracellular matrix (ECM) component. We found that by tuning the initial protonation state of carboxylic acid residues of HA in a covalently crosslinked hydrogel modulate BMP-2 release at physiological pH by minimizing the electrostatic repulsion and maximizing the Van der Waals interactions. At neutral pH, BMP-2 release is primarily governed by Fickian diffusion, whereas at acidic pH both diffusion and electrostatic interactions between HA and BMP-2 become important as confirmed by molecular dynamics simulations. Our results were also validated in an in vivo rat ectopic model with rhBMP-2 loaded hydrogels, which demonstrated superior bone formation with acidic hydrogel as compared to the neutral counterpart. We believe this study provides new insight on growth factor stabilization and highlights the therapeutic potential of engineered matrices for rhBMP-2 delivery and may help to curtail the adverse side effects associated with the high dose of the growth factor.

Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds.

BackgroundSuccessful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity.MethodsIn this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w).ResultsEvaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group.ConclusionThese results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration.

Human Gingival Fibroblast: Single Colonies vs. Heterogeneous Culture, Characterization and Osteogenic Differentiation

Cells with mesenchymal stem cell (MSC) properties were successfully isolated and characterized from different dental tissues. Amongst these cells are the human gingival fibroblast cells (GFs). Their use in tissue engineering is promising. However, choosing the right cellular population is one of several factors that are necessary for a successful tissue engineering approach. In order to better choose which population of cells to use, we isolated the GFs single colonies. We identified them, osteogenically induced them and compared them to the heterogeneous culture of these cells. Materials and methods: GFs cells were extracted from human gingival tissue; incubated to confluency. After which they were counted, serially diluted and seeded in 6 well plates. The cells were observed daily to locate the first formed colonies. Borosilicate cylinders were used to pick up the colonies. Flow cytometry was used to identify Stem cells surface markers to compare single colonies and heterogeneous cultures. The cells were then osteogenically induced for 21 days. The following assays were performed to compare the osteogenic potential between single colonies and heterogeneous cultures; Calcium assay, ALP/DNA specific activity, RT-qPCR for osteogenic related genes (OPN, OCN, ALP) and western blot analysis. Results: All the assays results were consistent in revealing an increased osteogenic differentiation potential of the heterogeneous culture of the GFs over the single colonies cultures. These results indicate that the heterogeneous cultures of GFs have a higher stem cell population and subsequent osteogenic differentiation potential than the single cell colonies’ cultures.

Synergistic effect of electrical conductivity and biomolecules on human meniscal cell attachment, growth, and proliferation in poly-ε-caprolactone nanocomposite scaffolds

Poly-ε-caprolactone (PCL)-based nanocomposite scaffolds with different concentrations of carbon nanofillers (carbon nanofibers (CNFs), nanographite, and exfoliated graphite) have been studied to investigate the effect of electrical conductivity and biomolecule supplementation for enhanced human meniscal cell attachment, growth, and proliferation. The incorporation of carbon nanofillers was found to improve the mechanical and electrical properties. CNF-based nanocomposite scaffolds showed the highest electrical conductivity with significant improvements in mechanical properties (more than 50% tensile strength increase than PCL with 10% (w/w) CNF). All nanocomposite scaffolds were subjected to cytotoxicity studies using primary meniscus cells. The nanocomposite scaffolds showing higher cell viability were selected and tested for meniscal cell attachment and proliferation assays such as total deoxyribonucleic acid content, extracellular matrix secretion, nuclear staining, and cell attachment studies using a scanning electron microscope. When an optimized combination of biomolecules is supplemented in the cell culture medium, a synergistic effect of the electrical conductivity and biomolecule combination is observed, especially in the case of highly conducting CNF (7.5% and 10% (w/w))-based nanocomposite scaffolds. Our findings suggest that electrically conductive scaffolds with optimized biomolecules in cell culture medium can potentially be used for successful human meniscal tissue engineering applications.

Femtosecond-Laser-Based 3D Printing for Tissue Engineering and Cell Biology Applications.

Fabrication of 3D cell scaffolds has gained tremendous attention in recent years because of its applications in tissue engineering and cell biology applications. The success of tissue engineering or cell interactions mainly depends on the fabrication of well-defined microstructures, which ought to be biocompatible for cell proliferation. Femtosecond-laser-based 3D printing is one of the solution candidates that can be used to manufacture 3D tissue scaffolds through computer-aided design (CAD) which can be efficiently engineered to mimic the microenvironment of tissues. UV-based lithography has also been used for constructing the cellular scaffolds but the toxicity of UV light to the cells has prevented its application to the direct patterning of the cells in the scaffold. Although the mask-based lithography has provided a high resolution, it has only enabled 2D patterning not arbitrary 3D printing with design flexibility. Femtosecond-laser-based 3D printing is trending in the area of tissue engineering and cell biology applications due to the formation of well-defined micro- and submicrometer structures via visible and near-infrared (NIR) femtosecond laser pulses, followed by the fabrication of cell scaffold microstructures with a high precision. Laser direct writing and multiphoton polymerization are being used for fabricating the cell scaffolds, The implication of spatial light modulators in the interference lithography to generate the digital hologram will be the future prospective of mask-based lithography. Polyethylene glycol diacrylate (PEG-DA), ormocomp, pentaerythritol tetraacrylate (PETTA) have been fabricated through TPP to generate the cell scaffolds, whereas SU-8 was used to fabricate the microrobots for targeted drug delivery. Well-designed and precisely fabricated 3D cell scaffolds manufactured by femtosecond-laser-based 3D printing can be potentially used for studying cell migration, matrix invasion and nuclear stiffness to determine stage of cancer and will open broader horizons in the future in tissue engineering and biology applications.