Name: synthesis of graphene-hydroxyapatite nanocomposites for potential use in bone tissue engineering
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Dr. Sougata Ghosh acknowledges the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India, and Jawaharlal Nehru Centre for Advanced Scientific Research, India for funding under the Post-Doctoral Overseas Fellowship in Nano Science and Technology (Ref. JNC/AO/A.0610.1(4) 2019-2260 dated August 19, 2019). Dr. Sougata Ghosh acknowledges Kasetsart University, Bangkok, Thailand for a Post-Doctoral Fellowship, and funding under the Reinventing University Program (Ref. No. 6501.0207/10870 dated November 9, 2021). The authors would like to thank the Kostas Advanced Nano-Characterization Facility (KANCF) for assistance with the characterization experiments. KANCF is a shared multidisciplinary research and educational facility within the Kostas Research Institute (KRI) at Northeastern University.

1. Synthesis of nHAP by precipitation
<ol>
	<li>Synthesize the pristine nHAP using 50 mL of the reaction mixture containing 1 M Ca(NO3)2&#8729;4H2O and 0.67 M (NH4)H2PO4 followed by the dropwise addition of NH4OH (25%) to maintain a pH around 1018.</li>
	<li>Thereafter, agitate the reaction mixture by ultrasound irradiation (UI) for 30 min (500 W power and 20 kHz ultrasound frequency).</li>
	<li>Allow the resulting solution to mature for 120 h at room temperature until the white precipitate of nHAP settles out. Recover the nHAP by centrifugation at 1398 x g for 5 min at room temperature.</li>
	<li>Wash the precipitate with deionized (DI) water 3x and lyophilize for 48 h. Store the dry powder at 4 &#176;C.</li>
</ol>
2. Preparation of nHAP/GNR nanocomposites
NOTE: The following describes two approaches for fabricating nHAP/GNR (i.e., nHAP on GNR surfaces) and GNR/nHAP (GNR-coated nHAP) nanocomposites that represent two different spatial arrangements of nHAP and GNRs (Figure 1).
<ol>
	<li>Synthesis of nHAP/GNR
	<ol>
		<li>To prepare the nHAP/GNRs nanocomposite, use a co-functionalization strategy where nHAP can be synthesized and conjugated to GNRs simultaneously, as follows.</li>
		<li>Dissolve 5 mg of GNRs (Table of Materials) in a mixture of 1 M calcium nitrate tetrahydrate [Ca(NO3)2&#183;4H2O] and 0.67 M diammonium hydrogen phosphate [(NH4)2HPO4] to a 50 mL final volume19.</li>
		<li>During this reaction, add 25% of NH4OH dropwise to maintain the pH at ~10. Agitate the resulting mixture by UI for 30 min.</li>
		<li>After completion of the reaction, leave the solution undisturbed for 120 h at room temperature until maturation.</li>
		<li>Observe the formation of a gelatinous precipitate of nHAP which coats the GNRs, following which a white precipitate of nHAP/GNRs settles.</li>
		<li>Wash the precipitate 3x by centrifugation at 1398 x g for 5 min at room temperature followed by re-dispersion in DI water.</li>
		<li>Lyophilize the recovered washed precipitate for 48 h. Store the dry powder at 4 &#176;C.</li>
		<li>Use pristine nHAP and GNRs as control samples.</li>
	</ol>
	</li>
	<li>Synthesis of GNR/nHAP nanocomposite
	<ol>
		<li>Suspend commercially available nHAP at a concentration of 5 mg/mL in 50 mL of DI water supplemented with 5 mg of GNRs.</li>
		<li>Agitate the resulting mixture by UI for 30 min and thereafter leave the mixture undisturbed for 120 h at room temperature.</li>
		<li>After maturation, recover the white precipitate of the resulting GNR/nHAP by centrifugation at 1398 x g for 5 min at room temperature.</li>
		<li>Wash the sample 3x using DI water, lyophilize it for 48 h, and store the dry powder at 4 &#176;C for further use.</li>
	</ol>
	</li>
</ol>
3. Characterization of nHAP, nHAP/GNR, and GNR/nHAP
<ol>
	<li>Use a high-resolution transmission electron microscope (HRTEM) (see Table of Materials) to characterize the morphology and size of the nanocomposites11.</li>
	<li>Analyze the elemental composition of the nanocomposites employing energy dispersive spectroscopy (EDS) and perform elemental mapping using the scanning transmission electron microscope&#160;(STEM)11.</li>
	<li>Perform Fourier transform infrared (FTIR) spectroscopy for the neat samples at wavenumbers of 500-4000 cm&#8722;1 to analyze the chemical groups in the nanocomposite16.</li>
	<li>Perform powder X-ray diffraction (XRD) analysis of the as-synthesized nHAP using an X-ray wavelength of 1.5406 &#197;, current and voltage settings of 40 mA and 40 kV, respectively, and 2&#952; ranging from 20&#176; to 90&#176;.</li>
	<li>Evaluate the percent loading of GNR in the nanocomposite using thermogravimetric analysis (TGA) by heating the samples from room temperature to 1000 &#176;C at a rate of 10 &#176;C/min under nitrogen flow.</li>
</ol>

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The authors have no conflicts of interest.

Although various metals, polymers, ceramics, and their combinations have been researched as orthopedic implants and fixation accessories, HAP is considered to be one of the most preferable materials due to its chemical similarity to the bone itself and consequent high cytocompatibility20,21,22. In this study, the orientation of HAP was varied, which can have a significant impact on its unique properties, such as promotion of ost...

Graphene has sheet-like two-dimensional structures composed of sp-hybridized carbon. Several other allotropes can be attributed to the extended honeycomb network of graphene (e.g., the stacking of graphene sheets forms 3D graphite while rolling off the same material results in the formation of 1D nanotubes1). Likewise, 0D fullerenes are formed due to wrapping2. Graphene has attractive physicochemical and optoelectronic properties that include an ambipolar field-effect and a quantum Hall effect at room temperature3,4. Detection of single-molecule adsorption events and extremely high carrier mobility add to the attractive attributes of graphene5,6. Further, graphene nanoribbons (GNRs) with narrow widths and a large mean free path, low resistivity with a high current density, and high electron mobility are considered promising interconnecting materials7. Hence, GNRs are being explored for applications in a myriad of devices, and more recently in nanomedicine, particularly tissue engineering and drug delivery8.
Among various traumatic ailments, bone injuries are considered one of the most challenging due to difficulties in stabilizing the fracture, regeneration and replacement with new bone, resisting infection, and re-aligning bone non-unions9,10. Surgical procedures remain the only alternative for femoral shaft fractures. It should be noted that almost $52 million is spent every year on treating bone injuries in Central America and Europe11.
Bioactive scaffolds for bone tissue engineering applications can be more effective by incorporating nano-hydroxyapatite (nHAP), as they resemble the micro and nano architectural properties of the bone itself12. HAP, chemically represented as Ca10(PO4)6(OH)2 with a Ca/P molar ratio of 1.67, is the most preferred for biomedical applications, particularly for treating periodontal defects, the substitution of hard tissues, and fabricating implants for orthopedic surgeries13,14. Thus, the fabrication of nHAP-based biomaterials reinforced with GNRs can possess superior biocompatibility and may be advantageous due to their ability to promote osseointegration and be osteoconductive15,16. Such hybrid composite scaffolds can preserve biological properties such as cell adherence, spreading, proliferation, and differentiation17. Herein, we report the fabrication of two new nanocomposites for bone tissue engineering by rationally altering the spatial arrangement of nHAP and GNRs as illustrated in Figure 1. The chemical and structural properties of the two different nHAP-GNRs arrangements were evaluated here.

<table><tbody><tr><td>Ammonium phosphate monobasic</td><td>Sigma-Aldrich</td><td>216003-100G</td><td>Synthesis</td></tr><tr><td>Calcium nitrate tetrahydrate</td><td>Sigma-Aldrich</td><td>237124</td><td>Synthesis</td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>EBA 200S</td><td>Recovery</td></tr><tr><td>Fourier transform infrared spectrometer</td><td>Brucker</td><td>Vertex 70</td><td>Characterization</td></tr><tr><td>Graphene nanoribbon</td><td>Sigma-Aldrich</td><td>922714</td><td>Synthesis</td></tr><tr><td>High resolution transmission electron microscope</td><td>Thermo Fisher Scientific</td><td>Themis Titan 300</td><td>Characterization</td></tr><tr><td>Magnetic stirrer</td><td>IKA</td><td>C-MAG HS7 S68</td><td>Functionalization</td></tr><tr><td>Micropipettes</td><td>TreffLab</td><td>06H35687</td><td>Reagent preparation</td></tr><tr><td>pH meter</td><td>Eutech pH5+</td><td>ECPH503PLUSK</td><td>Reagent preparation</td></tr><tr><td>Thermogravimetric analyzer</td><td>TA Instruments</td><td>SDT Q600</td><td>Characterization</td></tr><tr><td>Ultrasonic bath</td><td>Bandelin</td><td>DT100</td><td>Functionalization</td></tr><tr><td>Universal Oven</td><td>Memmert</td><td>UF55</td><td>Functionalization</td></tr><tr><td>Weighing balance</td><td>Precisa</td><td>XB220A</td><td>Reagent preparation</td></tr><tr><td>X-ray diffractometer</td><td>Brucker</td><td>D8-Advanced</td><td>Characterization</td></tr></tbody></table>

synthesis of graphene-hydroxyapatite nanocomposites for potential use in bone tissue engineering

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been fabricated with hydroxyapatite to facilitate cell adherence, proliferation, and osteogenesis. In this study, hybrid nanocomposites were successfully developed using graphene nanoribbons (GNRs) and nanoparticles of hydroxyapatite (nHAPs), that when employed in bioactive scaffolds may potentially improve bone tissue regeneration. These nanostructures can be biocompatible. Here, two approaches were used for preparing the novel materials. In one approach, a co-functionalization strategy was used where nHAP was synthesized and conjugated to GNRs simultaneously, resulting in nanohybrids of nHAP on GNR surfaces (denoted as nHAP/GNR). High-resolution transmission electron microscopy (HRTEM) confirmed that the nHAP/GNR composite is comprised of slender, thin structures of GNRs (maximum length of 1.8 &#181;m) with discrete patches (150-250 nm) of needle-like nHAP (40-50 nm in length). In the other approach, commercially available nHAP was conjugated with GNRs forming GNR-coated nHAP (denoted as GNR/nHAP) (i.e., with an opposite orientation relative to the nHAP/GNR nanohybrid). The nanohybrid formed using the latter method exhibited nHAP nanospheres with a diameter ranging from 50 nm to 70 nm covered with a network of GNRs on the surface. Energy dispersive spectra, elemental mapping, and Fourier transform infrared (FTIR) spectra confirmed the successful integration of nHAP and GNRs in both nanohybrids. Thermogravimetric analysis (TGA) indicated that the loss at elevated heating temperatures due to the presence of GNRs was 0.5% and 0.98% for GNR/nHAP and nHAP/GNR, respectively. The nHAP-GNR nanohybrids with opposite orientations represent significant materials for use in bioactive scaffolds to potentially promote cellular functions for improving bone tissue engineering applications.

HRTEM analysis 
Individually, GNRs were slender bamboo-like structures with some bends at some distance as observed in Figure 2. The longest GNR was 1.841 &#181;m while the smallest bent GNR was 497 nm. The nanoribbons often showed a visible variation in width that might be attributed to twisting to form helical configurations in many places. Such unidirectional alignment of GNRs may help to obtain attractive features such as magnetic properties, conductivity, or heat t...

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Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

Novel nanocomposites of graphene nanoribbons and hydroxyapatite nanoparticles were prepared using solution-phase synthesis. These hybrids when employed in bioactive scaffolds can exhibit potential applications in tissue engineering and bone regeneration.

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been ...

synthesis-graphene-hydroxyapatite-nanocomposites-for-potential-use

Research

JoVE Journal

Bioengineering

3.6K Views.  Kasetsart University. Dit werk verklaart de strategie om nieuwe composieten van nanohydroxyapatiet en grafeen nanoribbons met tegengestelde oriëntaties te fabriceren. Deze biomaterialen zijn belangrijk voor het ontwikkelen van botweefsel engineering steigers. Dit is een eenvoudige eenpotsynthese.Het is een snelle, effectieve en economische methode. Dit werk is uiterst relevant in de context van de behandeling van botletsels en bijbehorende aandoeningen waarbij meer botweefselregeneratie een snelle genezing...

Video: Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Analyzing Cell-Seeded Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then&#160;assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;s modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived&#160;scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Author Spotlight: Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering 

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

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