In Vitro and In Vivo Evaluation of 3D Printed Poly(Ethylene Glycol) Dimethacrylate-Based Photocurable Hydrogel Platform for Bone Tissue Engineering.

This study focuses to develop a unique hybrid hydrogel bioink formulation that incorporates poly(ethylene glycol) dimethacrylate (PEGDMA), gelatin (Gel), and methylcellulose (MC). This formulation achieves the necessary viscosity for extrusion-based three-dimensional (3D) printing of scaffolds intended for bone regeneration. After thorough optimization of the hybrid bioink system with Gel, three distinct scaffold groups are investigated in vitro: 0%, 3%, and 6% (w/v) Gel. These scaffold groups are examined for their morphology, mechanical strength, biodegradation, in vitro cell proliferation and differentiation, and in vivo bone formation using a rat cranial defect model. Among these scaffold compositions, the 3% Gel scaffold exhibits the most favorable characteristics, prompting further evaluation as a rat mesenchymal stem cell (rMSC) carrier in a critical-size cranial defect within a Lewis rat model. The compressive strength of all three scaffold groups range between 1 and 2 MPa. Notably, the inclusion of Gel in the scaffolds leads to enhanced bioactivity and cell adhesion. The Gel-containing scaffolds notably amplify osteogenic differentiation, as evidenced by alkaline phosphatase (ALP) and Western blot analyses. The in vivo results, as depicted by microcomputed tomography, showcase augmented osteogenesis within cell-seeded scaffolds, thus validating this innovative PEGDMA-based scaffold system as a promising candidate for cranial bone defect healing.

Full factorial design of experiment-based and response surface methodology approach for evaluating variation in uniaxial compressive mechanical properties, and biocompatibility of photocurable PEGDMA-based scaffolds.

The goal of this study is to fabricate biocompatible and minimally invasive bone tissue engineering scaffolds that allowin situphotocuring and further investigate the effect on the mechanical properties of the scaffold due to the prevailing conditions around defect sites, such as the shift in pH from the physiological environment and swelling due to accumulation of fluids during inflammation. A novel approach of incorporating a general full factorial design of experiment (DOE) model to study the effect of the local environment of the tissue defect on the mechanical properties of these injectable and photocurable scaffolds has been formulated. Moreover, the cross-interaction between factors, such as pH and immersion time, was studied as an effect on the response variable. This study encompasses the fabrication and uniaxial mechanical testing of polyethylene glycol dimethacrylate (PEGDMA) scaffolds for injectable tissue engineering applications, along with the loss in weight of the scaffolds over 72 h in a varying pH environment that mimicsin vivoconditions around a defect. The DOE model was constructed with three factors: the combination of PEGDMA and nano-hydroxyapatite referred to as biopolymer blend, the pH of the buffer solution used for immersing the scaffolds, and the immersion time of the scaffolds in the buffer solution. The response variables recorded were compressive modulus, compressive strength, and the weight loss of the scaffolds over 72 h of immersion in phosphate-buffered saline at respective pH. The statistical model analysis provided adequate information in explaining a strong interaction of the factors on the response variables. Further, it revealed a significant cross-interaction between the factors. The factors such as the biopolymer blend and pH of the buffer solution significantly affected the response variables, compressive modulus and strength. At the same time, the immersion time had a strong effect on the loss in weight from the scaffolds over 72 h of soaking in the buffer solution. The biocompatibility study done using a set of fluorescent dyes for these tissue scaffolds highlighted an enhancement in the pre-osteoblasts (OB-6) cell attachment over time up to day 14. The representative fluorescent images revealed an increase in cell attachment activity over time. This study has opened a new horizon in optimizing the factors represented in the DOE model for tunable PEGDMA-based injectable scaffold systems with enhanced bioactivity.

Recent advances in organoid engineering: A comprehensive review.

Organoid, a 3D structure derived from various cell sources including progenitor and differentiated cells that self-organize through cell-cell and cell-matrix interactions to recapitulate the tissue/organ-specific architecture and function in vitro. The advancement of stem cell culture and the development of hydrogel-based extracellular matrices (ECM) have made it possible to derive self-assembled 3D tissue constructs like organoids. The ability to mimic the actual physiological conditions is the main advantage of organoids, reducing the excessive use of animal models and variability between animal models and humans. However, the complex microenvironment and complex cellular structure of organoids cannot be easily developed only using traditional cell biology. Therefore, several bioengineering approaches, including microfluidics, bioreactors, 3D bioprinting, and organoids-on-a-chip techniques, are extensively used to generate more physiologically relevant organoids. In this review, apart from organoid formation and self-assembly basics, the available bioengineering technologies are extensively discussed as solutions for traditional cell biology-oriented problems in organoid cultures. Also, the natural and synthetic hydrogel systems used in organoid cultures are discussed when necessary to highlight the significance of the stem cell microenvironment. The selected organoid models and their therapeutic applications in drug discovery and disease modeling are also presented.

FDA-approved bone grafts and bone graft substitute devices in bone regeneration.

To induce bone regeneration there is a complex cascade of growth factors. Growth factors such as recombinant BMP-2, BMP-7, and PDGF are FDA-approved therapies in bone regeneration. Although, BMP shows promising results as being an alternative to autograft, it also has its own downfalls. BMP-2 has many adverse effects such as inflammatory complications such as massive soft-tissue swelling that can compromise a patient's airway, ectopic bone formation, and tumor formation. BMP-2 may also be advantageous for patients not willing to give up smoking as it shows bone regeneration success with smokers. BMP-7 is no longer an option for bone regeneration as it has withdrawn off the market. PDGF-BB grafts in studies have shown PDGF had similar fusion rates to autologous grafts and fewer adverse effects. There is also an FDA-approved bioactive molecule for bone regeneration, a peptide P-15. P-15 was found to be effective, safe, and have similar outcomes to autograft at 2 years post-op for cervical radiculopathy due to cervical degenerative disc disease. Growth factors and bioactive molecules show some promising results in bone regeneration, although more research is needed to avoid their adverse effects and learn about the long-term effects of these therapies. There is a need of a bone regeneration method of similar quality of an autograft that is osteoconductive, osteoinductive, and osteogenic. This review covers all FDA-approved bone regeneration therapies such as the "gold standard" autografts, allografts, synthetic bone grafts, and the newer growth factors/bioactive molecules. It also covers international bone grafts not yet approved in the United States and upcoming technologies in bone grafts.

Fabrication of porous chitosan particles using a novel two-step porogen leaching and lyophilization method with the label-free multivariate spectral assessment of live adhered cells.

Porous chitosan (CS) particles were fabricated using a novel two-step technique that employed a porogen leaching phase followed by lyophilization or freeze-drying. Poly(ethylene glycol) (PEG) was mixed as a porogen in two different quantities with the CS solution before particle synthesis via coacervation. After the PEG leached out into deionized (DI) water at an elevated constant temperature, the final freeze-dried CS particles revealed surface features that resembled pore pockets. A three-dimensional (3D) culture of murine osteoblast cell line (OB-6) was seeded on these particles to analyze the effect of the porous structure on the cell activity, as compared to a control group with no added porogen. The results highlighted an enhancement in cell adhesion and proliferation on the two porous sample groups. A Raman spectroscopy-based label-free technique for live cell biomarker analysis was applied using multivariate spectral analysis. Results of the spectral analysis in the molecular fingerprint region corresponding to the Raman shift between 900 cm-1 and 1700 cm-1inferred inter-group variations. The bands at 1005 cm-1 and 1375 cm-1 were assigned to the live cell biomarkers phenylalanine and glycosaminoglycan, respectively, and were assessed during the multivariate spectral analysis. The corresponding score plot and loading information generated from the Principal Component Analysis (PCA) of the Raman spectrum at day 7 and day 14, pointed at inter-group spectral variations related to cell adhesion and proliferation between the two porous CS particle groups and the control.

Evaluation of the optimal dosage of BMP-9 through the comparison of bone regeneration induced by BMP-9 versus BMP-2 using an injectable microparticle embedded thermosensitive polymeric carrier in a rat cranial defect model.

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.

Osteogenic differentiation cues of the bone morphogenetic protein-9 (BMP-9) and its recent advances in bone tissue regeneration.

Bone regeneration using bioactive molecules and biocompatible materials is growing steadily with the advent of the new findings in cellular signaling. Bone Morphogenetic Protein (BMP)-9 is a considerably recent discovery from the BMP family that delivers numerous benefits in osteogenesis. The Smad cellular signaling pathway triggered by BMPs is often inhibited by Noggin. However, BMP-9 is resistant to Noggin, thus, facilitating a more robust cellular differentiation of osteoprogenitor cells into preosteoblasts and osteoblasts. This review encompasses a general understanding of the Smad signaling pathway activated by the BMP-9 ligand molecule with its specific receptors. The robust osteogenic cellular differentiation cue provided by BMP-9 has been reviewed from a bone regeneration perspective with several in vitro as well as in vivo studies reporting promising results for future research. The effect of the biomaterial, chosen in such studies as the scaffold or carrier matrix, on the activity of BMP-9 and subsequent bone regeneration has been highlighted in this review. The non-viral delivery technique for BMP-9 induced bone regeneration is a safer alternative to its viral counterpart. The recent advances in non-viral BMP-9 delivery have also highlighted the efficacy of the protein molecule at a low dosage. This opens a new horizon as a more efficient and safer alternative to BMP-2, which was prevalent among clinical trials; however, BMP-2 applications have reported its downsides during bone defect healing such as cystic bone formation.

Hydrogel-based 3D bioprinting: A comprehensive review on cell-laden hydrogels, bioink formulations, and future perspectives.

Hydrogel plays a vital role in cell-laden three dimensional (3D) bioprinting, whereas those hydrogels mimic the physical and biochemical characteristics of native extracellular matrix (ECM). The complex microenvironment of the ECM does not replicate from the traditional static microenvironment of the hydrogel, but the evolution of the 3D bioprinting facilitates to accommodate the dynamic modulation and spatial heterogeneity of the hydrogel system. Selection of hydrogel for 3D bioprinting depends on the printing techniques including microextrusion, inkjet, laser-assisted printing, and stereolithography. In this review, we specifically cover the 3D printable hydrogels where cells can be encapsulated without significant reduction in the cell viability. The recent research highlights of the most widely used hydrogel materials are elucidated in terms of stability of the hydrogel system, cross-linking method, support cell types and their post-printing cell viability. Also, the techniques used to improve the mechanical and biological properties of the hydrogels, such as adding various organic and inorganic materials and making microchannels, are discussed. Furthermore, the recent advances in vascularized tissue construct and scaffold-free bioprinting as a promising method for vascularization are covered in this review. The recent trends in four-dimensional (4D) bioprinting as a stimuli-responsive formation of new organs, and 3D bioprinting based organ-on-chip systems are also discussed.

Recent trends in the application of widely used natural and synthetic polymer nanocomposites in bone tissue regeneration.

The goal of a biomaterial is to support the bone tissue regeneration process at the defect site and eventually degrade in situ and get replaced with the newly generated bone tissue. Nanocomposite biomaterials are a relatively new class of materials that incorporate a biopolymeric and biodegradable matrix structure with bioactive and easily resorbable fillers which are nano-sized. This article is a review of a few polymeric nanocomposite biomaterials which are potential candidates for bone tissue regeneration. These nanocomposites have been broadly classified into two groups viz. natural and synthetic polymer based. Natural polymer-based nanocomposites include materials fabricated through reinforcement of nanoparticles and/or nanofibers in a natural polymer matrix. Several widely used natural biopolymers, such as chitosan (CS), collagen (Col), cellulose, silk fibroin (SF), alginate, and fucoidan, have been reviewed regarding their present investigation on the incorporation of nanomaterial, biocompatibility, and tissue regeneration. Synthetic polymer-based nanocomposites that have been covered in this review include polycaprolactone (PCL), poly (lactic-co-glycolic) acid (PLGA), polyethylene glycol (PEG), poly (lactic acid) (PLA), and polyurethane (PU) based nanocomposites. An array of nanofillers, such as nano hydroxyapatite (nHA), nano zirconia (nZr), nano silica (nSi), silver nano particles (AgNPs), nano titanium dioxide (nTiO2), graphene oxide (GO), that is used widely across the bone tissue regeneration research platform are included in this review with respect to their incorporation into a natural and/or synthetic polymer matrix. The influence of nanofillers on cell viability, both in vitro and in vivo, along with cytocompatibility and new tissue generation has been encompassed in this review. Moreover, nanocomposite material characterization using some commonly used analytical techniques, such as electron microscopy, spectroscopy, diffraction patterns etc., has been highlighted in this review. Biomaterial physical properties, such as pore size, porosity, particle size, and mechanical strength which strongly influences cell attachment, proliferation, and subsequent tissue growth has been covered in this review. This review has been sculptured around a case by case basis of current research that is being undertaken in the field of bone regeneration engineering. The nanofillers induced into the polymeric matrix render important properties, such as large surface area, improved mechanical strength as well as stability, improved cell adhesion, proliferation, and cell differentiation. The selection of nanocomposites is thus crucial in the analysis of viable treatment strategies for bone tissue regeneration for specific bone defects such as craniofacial defects. The effects of growth factor incorporation on the nanocomposite for controlling new bone generation are also important during the biomaterial design phase.

Enhanced cell functions on graphene oxide incorporated 3D printed polycaprolactone scaffolds.

For tissue engineering applications, a porous scaffold with an interconnected network is essential to facilitate the cell attachment and proliferation in a three dimensional (3D) structure. This study aimed to fabricate the scaffolds by an extrusion-based 3D printer using a blend of polycaprolactone (PCL), and graphene oxide (GO) as a favorable platform for bone tissue engineering. The mechanical properties, morphology, biocompatibility, and biological activities such as cell proliferation and differentiation were studied concerning the two different pore sizes; 400 µm, and 800 µm, and also with two different GO content; 0.1% (w/w) and 0.5% (w/w). The compressive strength of the scaffolds was not significantly changed due to the small amount of GO, but, as expected scaffolds with 400 µm pores showed a higher compressive modulus in comparison to the scaffolds with 800 µm pores. The data indicated that the cell attachment and proliferation were increased by adding a small amount of GO. According to the results, pore size did not play a significant role in cell proliferation and differentiation. Alkaline Phosphate (ALP) activity assay further confirmed that the GO increase the ALP activity and further Elemental analysis of Calcium and Phosphorous showed that the GO increased the mineralization compared to PCL only scaffolds. Western blot analysis showed the porous structure facilitate the secretion of bone morphogenic protein-2 (BMP-2) and osteopontin at both day 7 and 14 which galvanizes the osteogenic capability of PCL and PCL + GO scaffolds.

Thermoresponsive Injectable Microparticle-Gel Composites with Recombinant BMP-9 and VEGF Enhance Bone Formation in Rats.

Bone morphogenetic protein-9 (BMP-9) has been shown to be the most osteogenic BMP. Most of these experiments, however, involve an adenovirus-transfection strategy. Here, we used the scaffold-based strategy to study the bone forming ability of recombinant BMP-9 combined with vascular endothelial growth factor (VEGF). A robust, injectable, multicomponent-releasing scaffold in the form of a composite gel was developed by combining chitosan microparticles (MPs) with thermosensitive gel (MPs-gel). The MPs acted as the carriers for BMP-9 and the gel was loaded with VEGF. The developed gel consisted of hydrophobic chains of methyl cellulose (MC) and the cross-linked structures of alginate (Alg) and calcium. Gelation was achieved at physiological temperature and thus facilitated the injection and localization of MPs enabling an increased efficacy of incorporated growth factors at the target site. A release profile of incorporated growth factors over a two-week period showed higher release of VEGF at each time point compared to that of BMP-9. Human mesenchymal stem cells (hMSCs) encapsulated within the MPs-gel maintained their viability. BMP-9 enhanced the proliferation of hMSCs along the surface of MPs. Furthermore, BMP-9 potently induced the osteogenic differentiation of encapsulated hMSCs elucidated by the increased alkaline phosphatase (ALP) activity and the higher expression of ALP, collagen 1, and osteocalcin genes. In addition, in vivo experiments demonstrated that MPs-gel with the combination of BMP-9-VEGF could significantly enhance both subcutaneous and cranial bone formation (p < 0.05). Taken together, the results here strongly suggest that BMP-9-VEGF incorporated MPs-gel holds promise as an injectable bone tissue engineering platform.

Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate.

Chitosan microparticles were mixed with chitosan and carboxymethyl cellulose solution to achieve a good binding between the microparticles. Three different compositions of scaffolds were made by varying the calcium phosphate (CaP) amount: 0%, 10%, and 20%. Potassium chloride was used as salt, to make pores inside the scaffolds after leaching out when immersed in phosphate buffer saline (PBS). Compressive strength and compressive modulus of both non-porous (before leaching out), and porous (after leaching out) scaffolds were measured according to the ASTM standards. The highest compressive strength of 27 MPa was reported on 10% CaP scaffolds while 20% CaP scaffolds showed the lowest. The increasing CaP content reduces the compressive strength of the scaffolds. The highest wet state compressive strength was reported on 0% CaP scaffolds with 0.36 MPs and 0.40 MPa at day 1 and day 3 respectively. In vitro cell culture studies showed good cell adhesion and cell proliferation on 10% CaP scaffolds.

Comparative investigation of porous nano-hydroxyapaptite/chitosan, nano-zirconia/chitosan and novel nano-calcium zirconate/chitosan composite scaffolds for their potential applications in bone regeneration.

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; Ca10(PO4)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; ZrO2), and nano-calcium zirconate (nCZ; CaZrO3). 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 < 0.05), compared to CS alone scaffolds (28 KPa and 0.25 MPa) and were comparable among CS-nHA, CS-nZrO, and CS-nCZ scaffolds. Peak force quantitative nanomechanical mapping (PFQNM) using an atomic force microscope (AFM) showed that the Young's modulus of composite material was higher compared to only CS (p < 0.001), and the values were similar among the composite materials. One of the major issues in the use of Zr based bioceramic materials in bone tissue regeneration applications is their lower osteoblasts response. This study has shown that CS-nCZ supported higher proliferation of pre-osteoblasts compared to CS-nZrO and the spreading was more similar to that observed in CS-nHA scaffolds. Taken together, results show that the physical and biological properties, studied here, of CS composite with Zr based bio-ceramic was comparable with CS-nHA composite scaffolds and hence show the prospective of CS-nCZ for future bone tissue engineering applications.

Nano-scale characterization of nano-hydroxyapatite incorporated chitosan particles for bone repair.

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°â€¯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.

Injectable nanosilica-chitosan microparticles for bone regeneration applications.

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 < 0.05) in the expression of alkaline phosphatase by the cells growing on all sample groups compared to the control (C) groups at day 14. The morphological characterization of these microparticles through scanning electron microscopy showed that these microparticles were well suited to be used as the injectable scaffolds with perfectly spherical shape and size. The incorporation of silica nanopowder altered the nano-roughness of the microparticles as observed through atomic force microscopy scans with roughness values going down from C to S3. The results in this study, taken together, show the potential of chitosan-tripolyphosphate-silica nanopowder microparticles for improved bone regeneration applications.

Drug transport mechanisms and in vitro release kinetics of vancomycin encapsulated chitosan-alginate polyelectrolyte microparticles as a controlled drug delivery system.

In this study, chitosan-alginate polyelectrolyte microparticles containing the antibiotic, vancomycin chloride were prepared using the ionotropic gelation (coacervation) technique. In vitro release and drug transport mechanisms were studied concerning the chitosan only and alginate only microparticles as a control group. Further, the effect of porosity on the drug transport mechanism was also studied for chitosan-alginate mixed particles produced by lyophilizing in contrast to the air-dried non-porous particles. According to the in vitro release data, alginate only and chitosan only microparticles showed burst release and prolonged release respectively. Chitosan-alginate lyophilized microparticles showed the best-controlled release of vancomycin with the average release of 22µg per day for 14days. Also, when increasing alginate concentration there was no increase in the release rate of vancomycin. The release data of all the microparticles were treated with Ritger-Peppas, Higuchi, Peppas-Sahlin, zero-order, and first-order kinetic models. The best fit was observed with Peppas-Sahlin model, indicating the drug transport mechanism was controlled by both Fickian diffusion and case II relaxations. Also, Fickian diffusion dominates the drug transport mechanism of all air-dried samples during the study period. However, the Fickian contribution was gradually reducing with time. Porosity significantly effects the drug transport mechanism as case II relaxation dominates after day 10 of the lyophilized microparticles.

Reconstruction of Craniomaxillofacial Bone Defects Using Tissue-Engineering Strategies with Injectable and Non-Injectable Scaffolds.

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.

Injectable porous nano-hydroxyapatite/chitosan/tripolyphosphate scaffolds with improved compressive strength for bone regeneration.

In this study we have fabricated porous injectable spherical scaffolds using chitosan biopolymer, sodium tripolyphosphate (TPP) and nano-hydroxyapatite (nHA). TPP was primarily used as an ionic crosslinker to crosslink nHA/chitosan droplets. We hypothesized that incorporating nHA into chitosan could support osteoconduction by emulating the mineralized cortical bone structure, and improve the Ultimate Compressive Strength (UCS) of the scaffolds. We prepared chitosan solutions with 0.5%, 1% and 2% (w/v) nHA concentration and used simple coacervation and lyophilization techniques to obtain spherical scaffolds. Lyophilized spherical scaffolds had a mean diameter of 1.33mm (n=25). Further, portion from each group lyophilized scaffolds were soaked and dried to obtain Lyophilized Soaked and Dried (LSD) scaffolds. LSD scaffolds had a mean diameter of 0.93mm (n=25) which is promising property for the injectability. Scanning Electron Microscopy images showed porous surface morphology and interconnected pore structures inside the scaffolds. Lyophilized and LSD scaffolds had surface pores <10 and 2µm, respectively. 2% nHA/chitosan LSD scaffolds exhibited UCS of 8.59MPa compared to UCS of 2% nHA/chitosan lyophilized scaffolds at 3.93MPa. Standardize UCS values were 79.98MPa and 357MPa for 2% nHA/chitosan lyophilized and LSD particles respectively. One-way ANOVA results showed a significant increase (p<0.001) in UCS of 1% and 2% nHA/chitosan lyophilized scaffolds compared to 0% and 0.5% nHA/chitosan lyophilized scaffolds. Moreover, 2% nHA LSD scaffolds had significantly increased (p<0.005) their mean UCS by 120% compared to 2% nHA lyophilized scaffolds. In a drawback, all scaffolds have lost their mechanical properties by 95% on the 2nd day when fully immersed in phosphate buffered saline. Additionally live and dead cell assay showed no cytotoxicity and excellent osteoblast attachment to both lyophilized and LSD scaffolds at the end of 14th day of in vitro studies. 2% nHA/chitosan scaffolds showed higher osteoblast attachment than 0% nHA/chitosan scaffolds.

Fabrication and characterization of carboxymethyl cellulose novel microparticles for bone tissue engineering.

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 10days 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.

Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro.

The main aims of this manuscript are to: i) determine the effect of commonly used antibiotics to treat osteoarticular infections on osteoblast viability, ii) study the dual release of the growth factor (BMP-7) and antibiotics (vancomycin and cefazolin) from chitosan microparticles iii) demonstrate the bioactivity of the antibiotics released in vitro on Staphylococcus epidermidis. The novelty of this work is dual delivery of growth factor and antibiotic from the chitosan microparticles in a controlled manner without affecting their bioactivity. Cefazolin and vancomycin have different therapeutic concentrations for their action in vivo and therefore, two different concentrations of the drugs were used. Osteoblast cytotoxicity test concluded that cefazolin concentrations of 50 and 100µg/ml were found to have positive influence on osteoblast proliferation. A significant increase in osteoblast proliferation was observed in the presence of cefazolin and BMP-7 in comparison with BMP-7 alone group; indicating cefazolin might play a role in osteoblast proliferation. On the other hand, vancomycin concentration of 1000µg/ml was found to significantly reduce (p<0.01) osteoblast proliferation in comparison with controls. The microbial study indicated that cefazolin at a minimum concentration of 21.5µg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30µg/ml was found to inhibit ~80% bacterial growth.