Effects of bone surface topography and chemistry on macrophage polarization.

Surface structure plays a crucial role in determining cell behavior on biomaterials, influencing cell adhesion, proliferation, differentiation, as well as immune cells and macrophage polarization. While grooves and ridges stimulate M2 polarization and pits and bumps promote M1 polarization, these structures do not accurately mimic the real bone surface. Consequently, the impact of mimicking bone surface topography on macrophage polarization remains unknown. Understanding the synergistic sequential roles of M1 and M2 macrophages in osteoimmunomodulation is crucial for effective bone tissue engineering. Thus, exploring the impact of bone surface microstructure mimicking biomaterials on macrophage polarization is critical. In this study, we aimed to sequentially activate M1 and M2 macrophages using Poly-L-Lactic acid (PLA) membranes with bone surface topographical features mimicked through the soft lithography technique. To mimic the bone surface topography, a bovine femur was used as a model surface, and the membranes were further modified with collagen type-I and hydroxyapatite to mimic the bone surface microenvironment. To determine the effect of these biomaterials on macrophage polarization, we conducted experimental analysis that contained estimating cytokine release profiles and characterizing cell morphology. Our results demonstrated the potential of the hydroxyapatite-deposited bone surface-mimicked PLA membranes to trigger sequential and synergistic M1 and M2 macrophage polarizations, suggesting their ability to achieve osteoimmunomodulatory macrophage polarization for bone tissue engineering applications. Although further experimental studies are required to completely investigate the osteoimmunomodulatory effects of these biomaterials, our results provide valuable insights into the potential advantages of biomaterials that mimic the complex microenvironment of bone surfaces.

Morphology of Nanostructured Tantalum Oxide Controls Stem Cell Differentiation and Improves Corrosion Behavior.

Tantalum is receiving increasing attention in the biomedical field due to its biocompatible nature and superior mechanical properties. However, the bioinert nature of tantalum still poses a challenge and limits its integration into the bone tissue. To address these issues, we fabricated nanotubular (NT), nanocoral (NC), and nanodimple morphologies on tantalum surfaces via anodization. The size of these nanofeatures was engineered to be approximately 30 nm for all anodized samples. Thus, the influence of the anodized nanostructured morphology on the chemical and biological properties of tantalum was evaluated. The NT and NC samples exhibited higher surface roughness, surface energy, and hydrophilicity compared to the nonanodized samples. In addition, the NT samples exhibited the highest corrosion resistance among all of the investigated samples. Biological experiments indicated that NT and NC samples promoted human adipose tissue-derived mesenchymal stem cell (hADMSC) spreading and proliferation up to 5 days in vitro. ALP, COL1A1, and OSC gene expressions as well as calcium mineral synthesis were upregulated on the NT and NC samples in the second and third weeks in vitro. These findings highlight the significance of nanostructured feature morphology for anodized tantalum, where the NT morphology was shown to be a potential candidate for orthopedic applications.

A typical method for decellularization of plants as biomaterials.

Decellularization is a process by which cells are removed from tissues or organs, leaving behind the extracellular matrix (ECM) structure. This process has gained interest in the fields of tissue engineering and regenerative medicine as a way to prepare suitable scaffolds for tissue reconstruction. Although the initial efforts come with the animal tissues, this technique can also be applied to various plant tissues with simple modifications, as plant-derived biomaterials have the benefit of being biocompatible and serving as a safe, all-natural substitute for synthetic or animal originated materials. Additionally, plant-derived biomaterials may help cells grow and differentiate, creating a three-dimensional environment for tissue regeneration and repair. Here we demonstrate a general method for plant tissue decellularization, including already experienced approaches and techniques.•Exhibit the basic steps for plant decellularization, which may be applied to several other plant tissues.•The proposed approach may be optimized considering various intended uses.•Gives basic information for the determination of decellularization efficiency.

Emerging applications of 3D engineered constructs from plant seed extracts.

Seeds, by-products derived from various plants such as mango, quince, and apples, are considered waste, though they have emerging commercial potential, and have been used in biological, industrial, and physiological research. Seed-derived natural macromolecules- mainly polysaccharides, mucilage, gums, and cellulose-have physicochemical and structural diversification, giving the potential for forming gels, texturing, thickening, and providing interfacial adsorption. Seed-derived natural macromolecules have been widely used during the last few years in cell research and tissue engineering applications. Their widespread approachability and safety, high rate of biodegradability, biocompatibility, supporting cell proliferation, and extracellular matrix synthesis are the main properties making plant seed derivatives appropriate for use. The gel-forming ability of these derivatives gives them the capability of creating natural polymer-based scaffolds with the aptitude to resemble extracellular matrices (ECM). These ECM exhibit the high potential in scaffolds for tissue renewal. A deeper knowledge of the physicochemical characteristics of seed-derived mucilage and gum has been indicated as a key ingredient in several pharmaceutical preparations, but it has been remarkably utilized in nanomedicine for the last few years as a drug carrier for drug delivery, in gene therapy, and as scaffold components for tissue engineering purposes. Here, we afford up-to-date data about the different extracts from plant seeds-mainly mucilage and gum, we summarize the extraction techniques used to isolate these macromolecules, and we focus on their application in scaffold fabrication for tissue engineering purposes and regenerative medicine applications.

Surgical method for critical sized cranial defects in rat cranium.

Cranial tissue models are a widely used model to show the bone repair and the regeneration ability of candidate biomaterials for tissue engineering purposes. Until now, efficacy studies of different biomaterials for calvarial defect bone regeneration have been reported, generally in small animal models. This paper offers a versatile, reliable, and reproducible surgical method for creating a critical-sized cranial defect in rats including critical steps and tried-and-tested tips. The method proposed here,•Shows a general procedure for in vivo cranial models.•Provide an insight to restore bone tissue repair that may be used in combination with several tissue engineering strategies•Is a crucial technique that may guide in vivo bone tissue engineering.

Fast and versatile electrochemical approach for soft tissue decellularization.

Decellularization is one of a promising technique in the field of biomaterials based on the idea of using an acellular construct, here the organ / tissue itself, as a biocompatible and biological construct. In the decellularization process, the main objective is to preserve the structural and functional properties while removing living cells. In the current paper, we describe an electrochemical method for soft tissue decellularization at a specific voltages and time intervals, as well as further DNA, GAG, protein determinations, and histological examinations for the determination of decellularization efficacy. The approach proposed here, is:•Successful decellularization can be achieved by exposing the tissues to fewer chemicals than the traditional methods.•A facile and fast decellularization process long less than a day•An easy decellularization technique that may be applied to soft tissues.

Bone surface mimicked PDMS membranes stimulate osteoblasts and calcification of bone matrix.

Cellular microenvironments play a crucial role in cell behavior. In addition to the biochemical cues present in the microenvironments, biophysical and biomechanical properties on surfaces have an impact on cellular functionality and eventually cellular fate. Effects of surface topography on cell behavior are being studied extensively in the literature. However, these studies often try to replicate topographical features of tissue surfaces by using techniques such as chemical etching, photolithography, and electrospinning, which may result in the loss of crucial micro- and nano- features on the tissue surfaces such as bone. This study investigates the topographical effects of bone surface by transferring its surface features onto polydimethylsiloxane (PDMS) membranes using soft lithography from a bovine femur. Our results have shown that major features on bone surfaces were successfully transferred onto PDMS using soft lithography. Osteoblast proliferation and calcification of bone matrix have significantly increased along with osteoblast-specific differentiation and maturation markers such as osteocalcin (OSC), osterix (OSX), collagen type I alpha 1 chain (COL1A1), and alkaline phosphatase (ALP) on bone surface mimicked (BSM) PDMS membranes in addition to a unidirectional alignment of osteoblast cells compared to plain PDMS surfaces. This presented bone surface mimicking method can provide a versatile native-like platform for further investigation of intracellular pathways regarding osteoblast growth and differentiation.

Hollow microneedle array fabrication using a rational design to prevent skin clogging in transdermal drug delivery.

Microneedle (MN) technology is promising to replace hypodermic needles for practical use and painless drug delivery. However, the complex top-down fabrication process of functional MN arrays is a bottleneck that hinders their widespread use. Here, we fabricate the tapered hollow MN array using a unique bi-level-tip by combining strain-engineering and capillary self-assembly of carbon nanotube (CNT) microstructures. Strain-engineering facilitated by the offset pattern of the catalyst enables the growth of bent, bi-level CNT microstructures while capillary self-assembly helps in constituting the tapered geometry of MNs. The bottom-up fabrication that consists of only two standard photolithography steps and CNT growth to form the scaffold of MNs followed by a polymer (polyimide) reinforcement step to impart mechanical stiffness to MNs provides scalable and fewer processing steps. The tapered shape of the MN allows an 8 times smaller force to pierce and penetrate the skin compared to the straight MN. The liquid delivery rate of the bi-level-tip MN is measured to be 26% better than the flat tip MN of the same lumen size as its geometry reduces skin clogging effect at the needle tip. In addition, cytotoxicity tests verify that the polyimide reinforced CNT-MNs are biocompatible for future in vivo applications.

Decellularized Bone Extracellular Matrix-Coated Electrospun PBAT Microfibrous Membranes with Cell Instructive Ability and Improved Bone Tissue Forming Capacity.

Current approaches to develop bone tissue engineering scaffolds have some limitations and shortcomings. They mainly suffer from combining mechanical stability and bioactivity on the same platform. Synthetic polymers are able to produce mechanically stable sturctures with fibrous morphology when they are electrospun, however, they cannot exhibit bioactivity, which is crucial for tissue engineering and regenerative medicine. One current strategy to bring bioactivity in synthetic materials is to combine extracellular matrix (ECM)-sourced materials with biologically inert synthetic materials. ECM-sourced materials without any modifications are mechanically unstable; therefore, reinforcing them with mechanically stable platforms is indispensable. In order to overcome this bifacial problem, we have demonstrated that poly(butylene adipate-co-terephthalate) (PBAT) electrospun microfibrous membranes can be successfully modified with decellularized bone ECM to endow fibers with bioactive hydrogel and mimic natural micro-features of the native bone tissue. The developed structures have been shown to support osteogenesis, confirmed by histochemical staining and gene expression studies. Furthermore, ECM-coated PBAT fibers, when they were aligned, supplied an improved level of osteogenesis. The strategy demonstrated can be adapted to any other tissues, and the emerging microfibrous, mechanically stable, and bioactive materials can find implications in the specific fields of tissue engineering and regenerative medicine.

Plant-Based Scaffolds in Tissue Engineering.

A wide range of platforms has been developed for 3D culture of cells in vitro to aggregate and align cells to resemble in vivo conditions in order to enhance communication between cells and promote differentiation. The cellulose skeleton of plant tissue can serve as an attainable scaffold for mammalian cells after decellularization, which is advantageous when compared to synthetic polymers or animal-derived scaffolds. Adjustable variables to modify the physical and biochemical properties of the resulting scaffolds include the protocol for the sodium dodecyl sulfate (SDS)-based decellularization procedure, surface coatings for cell attachment, plant type for decellularization, differentiation media, and integrity and shape of the substrate. These tunable cellulose platforms can host a wide range of mammalian cell types from muscle to bone cells, as well as malignancies. Here, fundamentals and applications of decellularized plant-based scaffolds are discussed. These biocompatible, naturally perfused, tunable, and easily prepared decellularized scaffolds may allow eco-friendly manufacturing frameworks for application in tissue engineering and organs-on-a-chip.

A facile surface modification of poly(dimethylsiloxane) with amino acid conjugated self-assembled monolayers for enhanced osteoblast cell behavior.

Polydimethylsiloxane (PDMS) is a biocompatible synthetic polymer and used in various applications due to its low toxicity and tunable surface properties. However, PDMS does not have any chemical cues for cell binding. Plasma treatment, protein coating or surface modification with various molecules have been used to improve its surface characteristics. Still, these techniques are either last for a very limited time or have very complicated experimental procedures. In the present study, simple and one-step surface modification of PDMS is successfully accomplished by the preparation of hydrophilic and hydrophobic amino acid conjugated self-assembled monolayers (SAMs) for enhanced interactions at the cell-substrate interface. Synthesis of histidine and leucine conjugated (3-aminopropyl)-triethoxysilane (His-APTES and Leu-APTES) were confirmed with proton nuclear magnetic resonance spectroscopy (1H NMR) and optimum conditions for the modification of PDMS with SAMs were investigated by X-ray photoelectron spectroscopy (XPS) analysis, combined with water contact angle (WCA) measurements. Results indicated that both SAMs enhanced cellular behavior in vitro. Furthermore, hydrophilic His-APTES modification provides a superior environment for the osteoblast maturation with higher alkaline phosphatase activity and mineralization. As histidine, leucine, and functional groups of these SAMs are naturally found in biological systems, modification of PDMS with them increases its cell-substrate surface biomimetic properties. This study establishes a successful modification of PDMS for in vitro cell studies, offering a biomimetic and easy procedure for potential applications in microfluidics, cell-based therapies, or drug investigations.

Development of metformin chain extended polyurethane elastomers as bone regenerative films.

In this study, novel elastomeric biodegradable bone regenerative films were developed from metformin (Met) and polyurethane (PU). Metformin was selected due to its osteogenic properties and proper chemical structure to react with PU prepolymer. Metformin was integrated into PU macromolecular structure as chain extender after the synthesis of PU prepolymer via condensation polymerization of polycaprolactone diol and hexamethylene diisocyanate. Chemical, thermal, viscoelastic properties of PU-Met films where characterized and discussed in terms of structure-property relationships. PU-Met films had Tg value around -45 °C and showed superior viscoelastic properties under 1 Hz and 10 Hz tensile oscillation frequencies during dynamic mechanical analysis. On the 21st day of biodegradation studies, PU-Met films degraded 2.3 ±â€¯0.1% and 37.8 ±â€¯4.2% in oxidative and enzymatic media, respectively. Cell-material interactions of elastomeric films were investigated by proliferation (MTT assay), alkaline phosphatase activity (ALP), calcium depositions (Alizarin Red Quantification) and morphological evaluations (SEM). Presence of metformin in PU formulation increased MC3T3-E1 attachment, proliferation and calcium deposition.

Preparation of MC3T3-E1 cell sheets through short-term osteogenic medium application.

Cell sheet engineering is an emerging field based on the acquisition of cells together with their extracellular matrix (ECM) and is used not only in vitro but also in regeneration studies of various tissues in the clinic. Within this scope, wide variety of cell types have been investigated in terms of sheet formation and underlying mechanism. MC3T3-E1 is a mouse pre-osteoblast cell line that has greatly attracted researchers' attention for bone tissue engineering (BTE) applications thanks to its high proliferation and differentiation properties. The potential of MC3T3-E1 cells on sheet formation and the effects of culture conditions have not been investigated in detail. This study aims to examine the effects of growth and osteogenic medium on cell sheet formation of MC3T3-E1. As a result of this study; intact, ECM-rich, transferable cell sheets at the beginning of the mineralization phase of the differentiation process were obtained by using osteogenic medium. Hereafter, 3D tissue model can be constructed by stacking MC3T3 cell sheets in vitro. This 3D model can conveniently be used for the development of novel biomaterials and in vitro drug screening applications to reduce the need for animal experiments.

Cranial bone regeneration via BMP-2 encoding mesenchymal stem cells.

Cranial bone repair and regeneration via tissue engineering principles has attracted a great deal of interest from researchers during last decade. Here, within this study, 6 mm critical-sized bone defect regeneration via genetically modified mesenchymal stem cells (MSC) were monitored up to 4 months. Cranial bone repair and new bone formations were evaluated by histological staining and real time PCR analysis in five different groups including autograft and bone morphogenetic protein-2 (BMP-2) transfected MSC groups. Results presented here indicate a proper cranial regeneration in autograft groups and a prospering regeneration for hBMP-2 encoding mesenchymal stem cells.

Clinical applications of decellularized extracellular matrices for tissue engineering and regenerative medicine.

Decellularization is the process of removing the cellular components from tissues or organs. It is a promising technology for obtaining a biomaterial with a highly preserved extracellular matrix (ECM), which may also act as a biological scaffold for tissue engineering and regenerative therapies. Decellularized products are gaining clinical importance and market space due to their ease of standardized production, constant availability for grafting and mechanical or biochemical superiority against competing clinical options, yielding clinical results ahead of the ones with autografts in some applications. Current drawbacks and limitations of traditional treatments and clinical applications can be overcome by using decellularized or acellular matrices. Several companies are leading the market with versatile acellular products designed for diverse use in the reconstruction of tissues and organs. This review describes ECM-based decellularized and acellular products that are currently in use for different branches of clinic.

Isolation and characterization of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have drawn great interest in the field of regenerative medicine, for cell replacement, immunomodulatory, and gene therapies. It has been shown that these multipotent stromal cells can be isolated from tissues such as bone marrow, adipose tissue, trimester amniotic tissue, umbilical cord blood, and deciduous teeth and can be expanded in adherent culture. They have the capacity to differentiate into cells of the connective tissue lineages in vitro and contribute to tissue parenchyma in vivo. However, proper in vitro manipulation of MSCs is a key issue to reveal a potential therapeutic benefit following transplantation into the patients. This chapter summarizes some of the essential protocols and assays used at our laboratory for the isolation, culture, differentiation, and characterization of mesenchymal stem cells from the bone marrow and adipose tissue.