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Current bone cement systems often demand free radical or metal-related initiators and/or catalysts for the crosslinking process, which may cause serious toxicity to the human body. In addition, the resultant dense scaffolds may have a prolonged degradation time and are difficult for cells to infiltrate and form new tissue. In this study, we developed a porous “click” organic—inorganic nanohybrid (PO-click-ON) cement that crosslinks via metal-free biorthogonal click chemistry and forms porous structures mimicking the native bone tissue via particulate leaching. Strain-promoted click reaction enables fast and efficient crosslinking of polymer chains with the exclusion of any toxic initiator or catalyst. The resulting PO-click-ON implants supported exceptional in vitro stem cell adhesion and osteogenic differentiation with a large portion of stem cells infiltrated deep into the scaffolds. In vivo study using a rat cranial defect model demonstrated that the PO-click-ON system achieved outstanding cell adsorption, neovascularization, and bone formation. The porous click cement developed in this study serves as a promising platform with multifunctionality for bone and other tissue engineering applications.

Scaffold-free spheroids offer great potential as a direct supply of cells for bottom-up bone tissue engineering. However, the building of functional spheroids with both cells and bioactive signals remains challenging. Here, we engineered functional spheroids with mesenchymal stem cells (MSCs) and two-dimensional hetero-nano-layers (2DHNL) that consisted of black phosphorus (BP) and graphene oxide (GO) to create a 3D cell-instructive microenvironment for large defect bone repair. The effects of the engineered 2D materials on the proliferation, osteogenic differentiation of stem cells was evaluated in an in vitro 3D spheroidal microenvironment. Excellent in vivo support of osteogenesis of MSCs, neovascularization, and bone regeneration was achieved after transplanting these engineered spheroids into critical-sized rat calvarial defects. Further loading of osteogenic factor dexamethasone (DEX) on the 2DHNL showed outstanding in vivo osteogenic induction and bone regrowth without prior in vitro culture in osteogenic medium. The shortened overall culture time would be advantageous for clinical translation. These functional spheroids impregnated with engineered 2DHNL enabling stem cell and osteogenic factor co-delivery could be promising functional building blocks to provide cells and differential clues in an all-in-one system to create large tissues for time-effective in vivo bone repair.

Phosphorylated-oligo [poly(ethylene glycol)fumarate] (Pi-OPF) was combined with functionalized clays to facilitate the extrusion 3D printing of Pi-OPF. Acrylated montmorillonite (Ac-MMT) was synthesized for the covalent crosslinking of MMT with the Pi-OPF. The incorporation of Ac-MMT was observed to improve the rheological properties of Pi-OPF, enabling a high-fidelity extrusion printing. A well-dispersed exfoliated MMT phase was observed within the polymer matrix after the crosslinking. This leveraged improved mechanical properties of the Pi-OPF hydrogels evident through the compressive analysis. Additionally, a unique bioink combining chitosan methacrylate (ChiMA) and gelatin was developed with a primary goal of depositing the cells on the 3D printed Pi-OPF scaffolds for uniform cell distribution and for facilitating a spatial interaction between cells and Ac-MMT particles. This bioink was shown to support the encapsulation and proliferation of the printed pre-osteoblasts by the live/dead cell assay results. This excellent cell responses were unaltered when the cell laden was deposited on 3D printed Pi-OPF scaffolds. Furthermore, the spatial interaction between cells and Ac-MMT elicited improved osteoblast responses indicated by the spreading of encapsulated cells and higher intracellular alkaline phosphatase (ALP) expression. Taken together, the results of this study present the combinatorial application of 3D printing and bioprinting to achieve desirable biological responses through the interaction between cells and biomaterials.

Hydrogel scaffolds have attracted attention to develop cellular therapy and tissue engineering platforms for regenerative medicine applications. Among factors, local mechanical properties of scaffolds drive the functionalities of cell niche. Dynamic mechanical analysis (DMA), the standard method to characterize mechanical properties of hydrogels, restricts development in tissue engineering because the measurement provides a single elasticity value for the sample, requires direct contact, and represents a destructive evaluation preventing longitudinal studies on the same sample. We propose a novel technique, acoustic force elastography microscopy (AFEM), to evaluate elastic properties of tissue engineering scaffolds.AFEM can resolve localized and two-dimensional (2D) elastic properties of both transparent and opaque materials with advantages of being non-contact and non-destructive. Gelatin hydrogels, neat synthetic oligo[poly(ethylene glycol)fumarate] (OPF) scaffolds, OPF hydroxyapatite nanocomposite scaffolds and ex vivo biological tissue were examined with AFEM to evaluate the elastic modulus. These measurements of Young's modulus range from approximately 2 kPa to over 100 kPa were evaluated and are in good agreement with finite element simulations, surface wave measurements, and DMA tests.The AFEM can resolve localized and 2D elastic properties of hydrogels, scaffolds and thin biological tissues. These materials can either be transparent or non-transparent and their evaluation can be done in a non-contact and non-destructive manner, thereby facilitating longitudinal evaluation.AFEM is a promising technique to quantify elastic properties of scaffolds for tissue engineering and will be applied to provide new insights for exploring elastic changes of cell-laden scaffolds for tissue engineering and material science.

Three-dimensional (3D) printing is a promising technology for tissue engineering. However, 3D-printing methods are limited in their ability to produce desired microscale features or electrochemical properties in support of robust cell adhesion, proliferation, and differentiation. This study addresses this deficiency by proposing an integrated, one-step, method to increase the cytocompatibility of 3D-printed scaffolds through functionalization leveraging conductive carbon nanotubes (CNTs). To this end, CNTs were first sonicated with water-soluble single-stranded deoxyribonucleic acid (ssDNA) to generate a negatively charged ssDNA@CNT nano-complex. Concomitantly, 3D-printed poly(propylene fumarate) (PPF) scaffolds were ammonolyzed to introduce free amine groups, which can take on a positive surface charge in water. The ssDNA@CNT nano-complex was then applied to 3D-printed scaffolds through a simple one-step coating utilizing electric-static force. This fast and facile functionalization step resulted in a homogenous and non-toxic coating of CNTs to the surface, which significantly improved the adhesion, proliferation, and differentiation of pre-osteoblast cells. In addition, the CNT based conductive coating layer enabled modulation of cell behavior through electrical stimuli (ES) leading to cellular proliferation and osteogenic gene marker expression, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Collectively, these data provide the foundation for a one-step functionalization method for simple, fast, and effective functionalization of 3D printed scaffolds that support enhanced cell adhesion, proliferation, and differentiation, especially when employed in conjunction with ES. STATEMENT OF SIGNIFICANCE: Three-dimensional (3D) printing is a promising technology for tissue engineering. However, 3D-printing methods have limited ability to produce desired features or electrochemical properties in support of robust cell behavior. To address this deficiency, the current study proposed an integrated, one-step method to increase the cytocompatibility of 3D-printed scaffolds through functionalization leveraging conductive carbon nanotubes (CNTs). This fast and facile functionalization resulted in a homogenous and non-toxic coating of CNTs to the surface, which significantly improved the adhesion, proliferation, and differentiation of cells on the 3D-printed scaffolds.

In situ cell recruitment is a promising regenerative medicine strategy with the purpose of tissue regeneration without stem cell transplantation. This chemotaxis-based strategy is aimed at ensuring a restorative environment through the release of chemokines that promote site-specific migration of healing cell populations. Stromal cell-derived factor-1α (SDF-1α) is a critical chemokine that can regulate the migration of mesenchymal stem cells (MSCs). Accordingly, here, SDF-1α-loaded microporous oligo[poly(ethylene glycol) fumarate]/bis[2-(methacryloyloxy)ethyl] phosphate composites (SDF-1α/OPF/BP) were engineered and probed. SDF-1α/OPF/BP composites were loaded with escalating SDF-1α concentrations, namely, 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml, and were cocultured with MSC. Scratching assay, Transwell assay, and three-dimensional migration model were utilized to assess the migration response of MSCs. Immunofluorescence staining of Runx2 and osteopontin (OPN), ELISA assay of osteocalcin (OCN) and alkaline phosphatase (ALP), and Alizarin Red S staining were conducted to assess the osteogenesis of MSCs. All SDF-1α/OPF/BP composites engendered a release of SDF-1α (>80%) during the first four days. SDF-1α released from the composites significantly promoted migration and osteogenic differentiation of MSCs documented by upregulated expression of osteogenic-related proteins, ALP, Runx2, OCN, and OPN. SDF-1α at 100 ng/ml was optimal for enhanced migration and osteogenic proficiency. Thus, designed SDF-1α/OPF/BP composites were competent in promoting the homing and osteogenesis of MSCs and thus offer a promising bioactive scaffold candidate for on-demand bone tissue regeneration.

Phosphorene, also known as black phosphorus (BP), is a two-dimensional (2D) material that has gained significant attention in several areas of current research. Its unique properties such as outstanding surface activity, an adjustable bandgap width, favorable on/off current ratios, infrared-light responsiveness, good biocompatibility, and fast biodegradation differentiate this material from other two-dimensional materials. The application of BP in the biomedical field has been rapidly emerging over the past few years. This article aimed to provide a comprehensive review of the recent progress on the unique properties and extensive medical applications for BP in bone, nerve, skin, kidney, cancer, and biosensing related treatment. The details of applications of BP in these fields were summarized and discussed.

Injectable polymers have attracted intensive attention in tissue engineering and drug delivery applications. Current injectable polymer systems often require free-radical or heavy-metal initiators and catalysts for the crosslinking process, which may be extremely toxic to the human body. Here, we report a novel polyhedral oligomeric silsesquioxane (POSS) based strain-promoted alkyne-azide cycloaddition (SPAAC) "click" organic-inorganic nanohybrids (click-ON) system that can be click-crosslinked without any toxic initiators or catalysts. The click-ON scaffolds supported excellent adhesion, proliferation, and osteogenesis of stem cells. In vivo evaluation using a rat cranial defect model showed outstanding bone formation with minimum cytotoxicity. Essential osteogenic alkaline phosphatase (ALP) and vascular CD31 marker expression were detected on the defect site, indicating excellent support of in vivo osteogenesis and vascularization. Using salt leaching techniques, an injectable porous click-ON cement was developed to create porous structures and support better in vivo bone regeneration. Beyond defect filling, the click-ON cement also showed promising application for spinal fusion using rabbits as a model. Compared to the current clinically used poly (methyl methacrylate) (PMMA) cement, this click-ON cement showed great advantages of low heat generation, better biocompatibility and biodegradability, and thus has great potential for bone and related tissue engineering applications.

Injectable hydrogels have unique advantages for the repair of irregular tissue defects. In this study, we report a novel injectable carbon nanotube (CNT) and black phosphorus (BP) gel with enhanced mechanical strength, electrical conductivity, and continuous phosphate ion release for tissue engineering. The gel utilized biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) polymer as the cross-linking matrix, with the addition of cross-linkable CNT-poly(ethylene glycol)-acrylate (CNTpega) to grant mechanical support and electric conductivity. Two-dimensional (2D) black phosphorus nanosheets were also infused to aid in tissue regeneration through the steady release of phosphate that results from environmental oxidation of phosphorus in situ. This newly developed BP-CNTpega-gel was found to enhance the adhesion, proliferation, and osteogenic differentiation of MC3T3 preosteoblast cells. With electric stimulation, the osteogenesis of preosteoblast cells was further enhanced with elevated expression of several key osteogenic pathway genes. As monitored with X-ray imaging, the BP-CNTpega-gel demonstrated excellent in situ gelation and cross-linking to fill femur defects, vertebral body cavities, and posterolateral spinal fusion sites in the rabbit. Together, these results indicate that this newly developed injectable BP-CNTpega-gel owns promising potential for future bone and broad types of tissue engineering applications.

The nanomaterials research spectrum has seen the continuous emergence of two-dimensional (2D) materials over the years. These highly anisotropic and ultrathin materials have found special attention in developing biomedical platforms for therapeutic applications, biosensing, drug delivery, and regenerative medicine. Three-dimensional (3D) printing and bioprinting technologies have emerged as promising tools in medical applications. The convergence of 2D nanomaterials with 3D printing has extended the application dynamics of available biomaterials to 3D printable inks and bioinks. Furthermore, the unique properties of 2D nanomaterials have imparted multifunctionalities to 3D printed constructs applicable to several biomedical applications. 2D nanomaterials such as graphene and its derivatives have long been the interest of researchers working in this area. Beyond graphene, a range of emerging 2D nanomaterials, such as layered silicates, black phosphorus, transition metal dichalcogenides, transition metal oxides, hexagonal boron nitride, and MXenes, are being explored for the multitude of biomedical applications. Better understandings on both the local and systemic toxicity of these materials have also emerged over the years. This review focuses on state-of-art 3D fabrication and biofabrication of biomedical platforms facilitated by 2D nanomaterials, with the comprehensive summary of studies focusing on the toxicity of these materials. We highlight the dynamism added by 2D nanomaterials in the printing process and the functionality of printed constructs.

Bone morphogenetic proteins (BMPs) are well known as enhancers and facilitators of osteogenesis during bone regeneration. The use of recombinant BMP-2 (rhBMP-2) in bone defect healing has drawbacks, which has driven the scouting for alternatives, such as recombinant BMP-9 (rhBMP-9), to provide comparable new bone formation. However, the dosage of rhBMP-9 is quintessential for the facilitation of adequate bone defect healing. Therefore, this study has been designed to evaluate the optimal dosage of BMP-9 by comparing the bone defect healing induced by rhBMP-9 over rhBMP-2. The chitosan (CS) microparticles (MPs), coated with BMPs, were embedded in a thermoresponsive methylcellulose (MC) and calcium alginate (Alg) based injectable delivery system containing a dosage of either 0.5 μg or 1.5 μg of the respective rhBMP per bone defect. A 5 mm critical-sized cranial defect rat model has been used in this study, and bone tissues were harvested at eight weeks post-surgery. The standard tools for comparing the new bone regeneration included micro computerized tomography (micro-CT) and histological analysis. A novel perspective of analyzing the new bone quality and crystallinity was employed by using Raman spectroscopy, along with its elastic modulus quantified through Atomic Force Microscopy (AFM). Results showed that the rhBMP-9 administered at a dosage of 1.5 μg per bone defect, using this delivery system, can adequately facilitate the bone void filling with ample new bone mineralization and crystallinity as compared to rhBMP-2, thus approving the hypothesis for a viable rhBMP-2 alternative.

3D bioprinting is a promising new tissue restoration technique that enables the precise deposition of cells and growth factors in order to more closely mimic the structure and function of native organs. In this study, we report the development of a new bioink using oligo(poly(ethylene glycol) fumarate) (OPF), a photo-crosslinkable and biodegradable polymer, for 3D bioprinting. In addition to OPF, a small portion of gelatin was also incorporated into the bioink with to make it bio-printable. After immersion in the cell medium, gelatin was eluted away to create a bioprinted scaffold of pure OPF. Excellent cell viability, spreading, and long-term proliferation of encapsulated cells was observed using both bone and nerve cells as examples. These results demonstrate that OPF bioink has great potential in future 3D bioprinting applications that aim to replicate complex, layered tissues and/or organs.

Zirconium (Zr) based bioceramic nanoparticles, as the filler material to chitosan (CS), for the development of composite scaffolds are less studied compared to hydroxyapatite nanoparticles. This is predominantly due to the biological similarity of nano-hydroxyapatite (nHA; Ca(10)(PO(4))(6)(OH)(2)) with bone inorganic component. In this study, we compared the physical and biological properties of CS composite scaffolds hybridized with nHA, nano-zirconia (nZrO; ZrO(2)), and nano-calcium zirconate (nCZ; CaZrO(3)). For the first time in this study, the properties of CS-nCZ composite scaffolds have been reported. The porous composite scaffolds were developed using the freeze-drying technique. The compressive strength and modulus were in the range of 50–55 KPa and 0.75–0.95 MPa for composite scaffolds, significantly higher (p

In this study, injectable porous spherical particles were fabricated using chitosan (CS) biopolymer, sodium tripolyphosphate (TPP), and nano-hydroxyapatite (nHA). TPP was primarily used as an ionic crosslinker to crosslink 2% (w/v) CS droplets. 2% (w/v) nHA was used to prepare nHA incorporated particles. The surface morphological properties and nanomechanical properties such as topography, deformation, adhesion, and dissipation of CS particles with and without nHA were studied using contact mode and peakforce quantitative nanomechanical property mapping mode in atomic force microscopy. The nHA spots have higher density than CS which leads to higher forces acting on the probe tip and higher energy dissipation to lift the tip from nHA areas. The cumulative release data showed that about 87% of total BMP-2 encapsulated within the particles was released by third week of experiment period. Degradation study was conducted to understand how the particles degradation occurs in the presence of phosphate buffered saline with continues shaking in an incubator at 37° C. In addition, BMP-2 release from the 2% nHA/CS particles was studied over a three weeks period and found that BMP-2 release was governed by the simple diffusion rather than the degradation of particles.

This study was aimed at assessing the effects of silica nanopowder incorporation into chitosan-tripolyphosphate microparticles with the ultimate goal of improving their osteogenic properties. The microparticles were prepared by simple coacervation technique and silica nanopowder was added at 0% (C), 2.5% (S1), 5% (S2) and 10% (S3) (w/w) to chitosan. We observed that this simple incorporation of silica nanopowder improved the growth and proliferation of osteoblasts along the surface of the microparticles. In addition, the composite microparticles also showed the increased expression of alkaline phosphatase and osteoblast specific genes. We observed a significant increase ( p

In this study we developed carboxymethyl cellulose (CMC) microparticles through ionic crosslinking with the aqueous ion complex of zirconium (Zr) and further complexing with chitosan (CS) and determined the physio-chemical and biological properties of these novel microparticles. In order to assess the role of Zr, microparticles were prepared in 5% and 10% (w/v) zirconium tetrachloride solution. Scanning electron microscopy (SEM) with energy dispersive X-ray spectrometer (EDS) results showed that Zr was uniformly distributed on the surface of the microparticles as a result of which uniform groovy surface was obtained. We found that Zr enhances the surface roughness of the microparticles and stability studies showed that it also increases the stability of microparticles in phosphate buffered saline. The crosslinking of anionic CMC with cationic Zr and CS was confirmed by fourier transform infrared spectroscopy (FTIR) results. The response of murine pre-osteoblasts (OB-6) when cultured with microparticles was investigated. Live/dead cell assay showed that microparticles did not induce any cytotoxic effects as cells were attaching and proliferating on the well plate as well as along the surface of microparticles. In addition, SEM images showed that microparticles support the attachment of cells and they appeared to be directly interacting with the surface of microparticle. Within 10 days of culture most of the top surface of microparticles was covered with a layer of cells indicating that they were proliferating well throughout the surface of microparticles. We observed that Zr enhances the cell attachment and proliferation as more cells were present on microparticles with 10% Zr. These promising results show the potential applications of CMC-Zr microparticles in bone tissue engineering.

The present study is the first of its kind dealing with the development of a specific bioceramic which qualifies as a potential material in hard-tissue replacements. Specifically, we report the synthesis and evaluation of smart injectable calcium phosphate bone cement (CPC) which we believe will be suitable for various kinds of orthopedic and spinal-fusion applications. The smart nature of this next generation orthopedic implant is attained by incorporating piezoelectric barium titanate (BT) particles into monetite-based (dicalcium phosphate anhydrous, DCPA) CPC composition. The main goal is to take advantage of the piezoelectric properties of BT, as electromechanical effect plays a vital role in fracture healing at the defect site and bone integration with the implant. Furthermore, radiopacity of BT would help in easy detection of the CPC presence at the fracture site during surgery. Results reveal that BT addition favors important properties of bone cement such as good compressive strength, injectability, bioactivity, biocompatibility, and even washout resistance. Most importantly, the self-setting nature of the bone cements are not compromised with BT incorporation. The in vitro results confirm that the developed bone-cement abides by the standard orthopedic requirements making it apt for real-time prosthetic materials.

Engineering craniofacial bone tissues is challenging due to their complex structures. Current standard autografts and allografts have many drawbacks for craniofacial bone tissue reconstruction; including donor site morbidity and the ability to reinstate the aesthetic characteristics of the host tissue. To overcome these problems; tissue engineering and regenerative medicine strategies have been developed as a potential way to reconstruct damaged bone tissue. Different types of new biomaterials; including natural polymers; synthetic polymers and bioceramics; have emerged to treat these damaged craniofacial bone tissues in the form of injectable and non-injectable scaffolds; which are examined in this review. Injectable scaffolds can be considered a better approach to craniofacial tissue engineering as they can be inserted with minimally invasive surgery; thus protecting the aesthetic characteristics. In this review; we also focus on recent research innovations with different types of stem-cell sources harvested from oral tissue and growth factors used to develop craniofacial bone tissue-engineering strategies.