Cartilage regeneration with dual-drug-releasing injectable hydrogel/microparticle system: In vitro and in vivo study.

In this study, we developed an injectable in situ forming hydrogel/microparticle system consisting of two drugs, melatonin and methylprednisolone, to investigate the capability of the system for chondrogenesis in vitro and in vivo. The chemical, mechanical, and rheological properties of the hydrogel/microparticle were investigated. For in vitro evaluation, the adipose-derived stem cells might be mixed with hydrogel/microparticles, then cellular viability was analyzed by acridine orange/propidium iodide and 4',6-diamidino-2-phenylindole staining and also dimethylmethylene blue assay were conducted to find the amount of proteoglycan. The real-time polymerase chain reaction for aggrecan, sex-determining region Y-Box 9, collagen I (COL1), and COL2 gene expression was performed after 14 and 21 days. For evaluation of cartilage regeneration, the samples were implanted in rabbit knees with cartilaginous experimental defects. Defects were created in both knees of three groups of rabbits. Group 1 was the control with no injection, and Groups 2 and 3 were loaded with hydrogel/cell and hydrogel/microparticle/cell; respectively. Then, after 3 and 6 months, histological evaluations of the defected sites were carried out. The amount of glycosaminoglycans after 14 and 21 days increased significantly in hydrogels/microparticles loaded with cells. The expression of marker genes was also significant in hydrogels/microparticles loaded with cells. According to histology analysis, the hydrogels/microparticles loaded with cells showed the best cartilage regeneration. Overall, our study revealed that the developed injectable hydrogel/microparticle can be used for cartilage regeneration.

Synthesis and characterization of electroconductive hydrogels based on oxidized alginate and polypyrrole-grafted gelatin as tissue scaffolds.

Electroconductive biocompatible hydrogels with tunable properties have extensively been taken into account in tissue engineering applications due to their potential to provide suitable microenvironmental responses for the cells. In the present study, novel electroconductive hydrogels are designed and synthesized by reacting oxidized alginate with polypyrrole-grafted gelatin copolymer (PPy-g-gelatin) via formation of a Schiff-base linkage. The influence of the composition and the concentration of the components on the compressive modulus and functional performance of the hydrogels is investigated. The conductivity of the hydrogels measured by a two-probe method increased by increasing the level of polypyrrole-grafted gelatin, and a conductivity of 0.7753 S m-1 was exhibited by the hydrogel composed of 8% w/v polypyrrole-grafted gelatin (oxidized alginate:gelatin:polypyrrole-grafted gelatin; 30 : 35 : 35% v/v). The hydrogel compressive modulus was shown to be enhanced by increasing the total concentration of hydrogel. The characteristic features of the prepared hydrogels, including swelling ratio, volume fraction, cross-link density, and mesh size, are also studied and analyzed. Besides, the conductive hydrogels have a smaller mesh size and higher cross-link density than the non-conductive hydrogels. However, the hydrogels with high cross-link density, small mesh size, and large pore size presented higher electroconductivity as a result of easier movement of the ions throughout the hydrogel. These conductive hydrogels exhibited electrical conductivity and biodegradability with cell viability, implying potential as scaffolds for tissue engineering.

Improved cellular response on functionalized polypyrrole interfaces.

Neuroregeneration strategies involve multiple factors to stimulate nerve regeneration. Neural support with chemical and physical cues to optimize neural growth and replacing the lesion neuron and axons are crucial for designing neural scaffolds, which is a promising treatment approach. In this study, polypyrrole polymerization and its functionalization at the interface developed by glycine and gelatin for further optimization of cellular response. Nanofibrous scaffolds were fabricated by electrospinning of polyvinyl alcohol and chitosan solutions. The electrospun scaffolds were polymerized on the surface by pyrrole monomers to form an electroactive interface for further applications in neural tissue engineering. The polymerized polypyrrole showed a positive zeta potential value of 57.5 ± 5.46 mV. The in vitro and in vivo biocompatibility of the glycine and gelatin-functionalized polypyrrole-coated scaffolds were evaluated. No inflammatory cells were observed for the implanted scaffolds. Further, DAPI nucleus staining showed a superior cell attachment on the gelatin-functionalized polypyrrole-coated scaffolds. The topography and tuned positively charged polypyrrole interface with gelatin functionalization is expected to be particularly efficient physical and chemical simultaneous factors for promoting neural cell adhesion.

Microfluidic Directed Synthesis of Alginate Nanogels with Tunable Pore Size for Efficient Protein Delivery.

Alginate is a biopolymer with favorable pH-sensitive properties for oral delivery of peptides and proteins. However, conventional alginate nanogels have limitations such as low encapsulation efficiency because of drug leaching during bead preparation and burst release in high pH values. These shortcomings originate from large pore size of the nanogels. In this work, we proposed an on-chip hydrodynamic flow focusing approach for synthesis of alginate nanogels with adjustable pore size to achieve fine-tunable release profile of the encapsulated bioactive agents. It is demonstrated that the microstructure of nanogels can be controlled through adjusting flow ratio and mixing time directed on microfluidic platforms consisting of cross-junction microchannels. In this study, the average pore size of alginate nanogels (i.e., average molecular weight between cross-links, Mc) was related to synthesis parameters. Mc was calculated from equations based on equilibrium swelling theory and proposed methods to modify the theory for pH-sensitive nanogels. In the equations we derived, size and compactness of nanogels are key factors, which can be adjusted by controlling the flow ratio. It was found that increase in flow ratio increases the size of nanogels and decreases their compactness. The size of on-chip generated nanogels for flow ratio of 0.02-0.2 was measured to be in the range of 68-138 nm. Moreover, a method based on the Mie theory was implemented to estimate the aggregation number (Nagg) of polymer chains inside the nanogels as an indicator of compactness. According to the size and compactness results along with equations of modified swelling theory, Mc obtained to be in the range of 0.5-0.8 kDa. The proposed method could be considered as a promising approach for efficient polypeptides encapsulation and their sustained release.

Raspberry-like PLA/PGS biodegradable microparticles with urethane linkages: Unlocking tailored release of magnesium ions and oxygen for bone tissue engineering.

Designing biodegradable microparticles with finely controlled release properties for tissue engineering systems remains a significant scientific challenge. This study introduces a novel approach by fabricating urethane-linked PLA/PGS microparticles loaded with magnesium peroxide. The microparticles offer potential applications in bone tissue engineering due to their ability to provide a controlled release of oxygen and magnesium ions while maintaining physiological pH. The PGS pre-polymer was synthesized via polycondensation and characterized using FTIR, 1H NMR, and GPC. Microparticle morphology transformed from smooth to raspberry-like upon incorporation of PGS, as observed by SEM. Microparticle size was tuned by varying PGS and PLA concentrations. FTIR analysis confirmed the successful formation of urethane links within the microparticles. MgO2-loaded PLA/PGS microparticles exhibited sustained release of dissolved oxygen and magnesium ions for 21 days while maintaining physiological pH better than PLA microparticles. Cell viability assays confirmed microparticle cytocompatibility, and ALP and Alizarin red assays demonstrated their ability to induce osteogenic differentiation. These findings highlight the potential of pH-controlled MgO2-loaded microparticles as an effective system for bone tissue engineering. In conclusion, this study presents a novel approach to designing biodegradable microparticles with adjustable release properties for bone tissue engineering. The urethane-based MgO2-loaded microparticles provide controlled release of oxygen and magnesium ions and regulate the environment's pH, making them a promising system for bone tissue engineering applications.

CD44 targeted-chondroitin sulfate nanoparticles: Fine-tuning hydrophobic groups to enhance in vitro pH-responsiveness and in vivo efficacy for advanced breast cancer treatment.

The design of tumor-targeting nanoparticles with precisely controlled physical-biological properties may improve the delivery of chemotherapeutic agents. This study introduces pH-sensitive chondroitin sulfate-cholesterol (ChS-Chol) nano-assemblies for targeted intracellular doxorubicin (Dox) delivery in breast cancer treatment. Various ChS-Chol copolymers were synthesized, yielding self-assembling nanostructures with adjustable lipophilic content. In an aqueous environment, the ChS-Chol conjugates could form self-assembled nanostructures with a narrower size variation and a high negative potential. Moreover, the carriers would rapidly disassemble and release Dox in response to acidic pH. The in vitro cytotoxicity assay exhibited concentration-related anti-proliferation activity with Dox-loaded nanoparticles against 4T1, MCF-7, and MDA-MB-231 breast cancer cells. The nanoparticles demonstrated enhanced early apoptosis induction, efficient cellular uptake, and improved prevention of tumor cell proliferation compared to free Dox. In vivo results showcased significant tumor growth inhibition, underscoring the potential of these nanoparticle-based drug delivery systems for breast cancer therapy. The study emphasizes tailored nanocarrier design, leveraging pH-responsiveness and precise hydrophobic tuning to achieve targeted and potent therapeutic effects in the fight against breast cancer.

The immune-stealth polymeric coating on drug delivery nanocarriers: In vitro engineering and in vivo fate.

Although essential nanosystems such as nanoparticles and nanocarriers are desirable options for transporting various drug molecules into the biological environment, they rapidly remove from the circulatory system due to their interaction with multiple in vivo barriers, especially the immune barrier, which will result in their short-term effects. In order to improve their effectiveness and durability in the circulatory system, the polymer coatings can use to cover the surface of nanoparticles and nanocarriers to conceal them from the immune system. Due to their different properties (like charge, elasticity, and hydrophilicity/hydrophobicity), these coatings can improve drug delivery nanosystem durability and therapeutic applications. The mentioned coatings have different types and are divided into various categories, such as synthetic polymers, polysaccharides, and zwitterionic polymers. Each of these polymers has unique properties based on its category, origin, and chemical structure that make them suitable for producing stealth drug delivery nanocarriers. In this review article, we have tried to explain the importance of these diverse polymer coatings in determining the fate of drug nanocarriers and then introduced the different types of these coatings and, finally, described various methods that directly and indirectly analyze the nanocoatings to determine the stability of nanoparticles in the body.

Enhanced neural differentiation by applying electrical stimulation utilizing conductive and antioxidant alginate-polypyrrole/poly-l-lysine hydrogels.

The challenge of restoration from neurodegenerative disorder requires effective solutions. To enhance the healing efficiencies, scaffolds with antioxidant activities, electroconductivity, and versatile features to encourage neuronal differentiation are potentially useful. Herein, polypyrrole-alginate (Alg-PPy) copolymer was used to design antioxidant and electroconductive hydrogels through the chemical oxidation radical polymerization method. The hydrogels have antioxidant effects to combat oxidative stress in nerve damage thanks to the introduction of PPy. Additionally, poly-l-lysine (PLL) provided these hydrogels with a great differentiation ability of stem cells. The morphology, porosity, swelling ratio, antioxidant activity, rheological behavior, and conductive characteristics of these hydrogels were precisely adjusted by altering the amount of PPy. Characterization of hydrogels showed appropriate electrical conductivity and antioxidant activity for neural tissue applications. Cytocompatibility, live/dead assays, and Annexin V/PI staining by flow cytometry using P19 cells confirmed the excellent cytocompatibility and cell protective effect under ROS microenvironment of these hydrogels in both normal and oxidative conditions. The neural marker investigation in the induction of electrical impulses was assessed through RT-PCR and immunofluorescence assay, demonstrating the differentiation of P19 cells to neurons cultured in these scaffolds. In summary, the antioxidant and electroconductive Alg-PPy/PLL hydrogels demonstrated excellent potential as promising scaffolds for treating neurodegenerative disorders.

Redox-Sensitive multifunctional hyaluronic acid-based nanomicelles with Fine-controlled anticancer drug release.

A significant contributor to cancer-related death globally is metastatic breast cancer. To reduce death rates, tumor-specific penetration and triggered drug release are crucial. Herein, targeted intracellular doxorubicin (Dox) delivery system was effectively prepared based on redox-sensitive hyaluronic acid-palmitoyl (HA-ss-PA) copolymers. The amphiphilic copolymers self-assembled into nano and showed outstanding drug-loading capacities and encapsulation efficiency for Dox. Micelles were stable under physiological conditions, but they quickly disintegrated in the presence of a reducing agent. The intracellular location of the fluorescent probe rhodamine b demonstrated that HA-ss-PA micelles are an efficient approach for drug delivery in breast cancer cells. Based on flow cytometry and live/dead assay, observations indicated that micelles induce apoptosis in both MCF-7 and MDA-MB-231 cells. In vivo evaluation in tumor-bearing mice confirmed that HA-ss-PA micelles exhibited excellent tumor-targeting activity. These findings imply that redox-sensitive HA-ss-PA micelles are promising candidates for use as intracellular delivery systems for hydrophobic anti-cancer drugs.

Controlled release of resveratrol from a composite nanofibrous scaffold: Effect of resveratrol on antioxidant activity and osteogenic differentiation.

Biocompatibility, mechanical strength, and osteogenesis properties of three-dimensional scaffolds are critical for bone tissue engineering. In addition, reactive oxygen species accumulate around bone defects and limit the activities of surrounding cells and bone formation. Therefore, the presence of an antioxidant in a bone tissue scaffold is also essential to address this issue. This study aimed to evaluate a composite nanofibrous scaffold similar to the natural extracellular matrix with antioxidant and osteogenic properties. To this end, polylactic acid (PLA)/organophilic montmorillonite (OMMT)/resveratrol (RSV) nanofibers were fabricated using the electrospinning method and characterized. RSV was used as an antioxidant, which promotes osteogenic differentiation, and OMMT was used as a mineral phase to increase the mechanical strength and control the release of RSV. The scaffolds' antioxidant activity was measured using DPPH assay and found 83.75% for PLA/OMMT/RSV nanofibers. The mechanical strength was increased by adding OMMT to the neat PLA. The biocompatibility of the scaffolds was investigated using an MTT assay, and the results did not show any toxic effects on human adipose mesenchymal stem cells (hASCs). Moreover, the Live/Dead assay indicated the appropriate distribution of live cells after 5 days. Cell culture results displayed that hASCs could adhere and spread on the surface of composite nanofibers. Meanwhile, the level of alkaline phosphatase, osteocalcin, and osteopontin was increased for hASCs cultured on the PLA/OMMT/RSV nanofibrous scaffold. Therefore, this study concludes that the RSV-loaded composite nanofibers with antioxidant and osteogenesis properties and appropriate mechanical strength can be introduced for bone tissue regeneration applications.

Size-adjustable self-assembled nanoparticles through microfluidic platform promotes neuronal differentiation of mouse embryonic stem cells.

Neuronal differentiation from stem cells is one of the most potent therapeutic approaches for recovering neurological function in individuals with neurodegenerative disorders. Herein, an on-demand intracellular retinoic acid released nanoparticles with tunable size and accurately controlled physico-biological properties have been prepared for achieving efficient neuronal differentiation. The amphiphilic chitosan oligosaccharide-cholesterol copolymers were synthesized by varying cholesterol content and self-assembled into spherical micelle in a microfluidic chip with different flow rates. Notably, the results indicated that by increasing the lipophilicity of the chitosan chain as well as mixing rate, the size of micelles was decreased. Retinoic acid (RA) was efficiently encapsulated in the core of micelles. The retinoic acid-containing nanoparticles could escape lysosome, accumulate in the cytoplasm, and release payload with a sustained pattern. The cytotoxicity assay of free retinoic acid and retinoic acid-loaded formulations against P19 embryonic stem cells confirmed the desirable safety of micelles. The result obtained from the uptake study showed that internalization of micelles occurs predominantly via lipid-raft endocytosis in the presence of higher cholesterol content. Moreover, the intracellular RA release upregulated the expression levels of neuronal factors. The micelles described here offer a promising nanomedicine strategy for neuronal differentiation of stem cells.

Peroxide mediated oxygen delivery in cancer therapy.

Hypoxia is a serious obstacle in cancer treatment. The aberrant vascular network as well as the abnormal extracellular matrix arrangement results in formation of a hypoxic regions in tumors which show high resistance to the curing. Hypoxia makes the cancer treatment challengeable via two mechanisms; first and foremost, hypoxia changes the cell metabolism and leads the cells towards an aggressive and metastatic phenotype and second, hypoxia decreases the efficiency of the various cancer treatment modalities. Most of the cancer treatment methods including chemotherapy, radiotherapy, photodynamic therapy, sonodynamic therapy and immunotherapy are negatively affected by the oxygen deprivation. Therefore, the regional oxygenation is requisite to alleviate the negative impacts of the hypoxia on tumor cells and tumor therapy modalities. A great deal of effort has been put forth to resolve the problem of hypoxia in tumors. Peroxides have gained tremendous attention as oxygen generating components in cancer therapy. The concurrent loading of the peroxides and cancer treatment components into a single delivery system can bring about a multipurpose delivery system and substantially encourage the success of the cancer amelioration. In this review, we have tried to after the description of a relation between hypoxia and cancer treatment modalities, discuss the role of peroxides in tumor hyperoxygenation and cancer therapy success. Thereafter, we have summarized a number of vehicles for the delivery of the peroxide alone or in combination with other therapeutic components for cancer treatment.

Fyodorov-Zuev keratoprosthesis implantation: long-term results in patients with multiple failed corneal grafts.

BACKGROUND: The long term results of the Fyodorov-Zuev keratoprosthesis are presented for ten patients with repeated graft failures. METHODS: The study took place at Dr Khodadoust Eye Hospital. The Fyodorov-Zuev keratoprosthesis was implanted in ten patients with repeated graft failures, including seven patients with old trachoma, two patients with chemical injury, and a patient with thermal injury. The outcome measures were visual acuity, retention, and complications of the keratoprosthesis. RESULTS: Preoperative best-uncorrected visual acuity (BUVA) ranged from counting fingers to light perception (median: light perception). The keratoprosthesis dramatically improved vision in most patients. Patients retained BUVA of 20/200-20/50 in 50%, 20/60-20/100 in 30%, 20/200 in 10% and 20/400 in 10%. Overall, 90% had 1 year postoperative vision of 20/200 or better. 70% of cases had BUVA of 20/200 or better at the last follow-up (average 52 months). Of ten keratoprosthesis implanted, three have been removed, and as a result, retention rate was 70% during average follow-up of 52 months .The most common postoperative complications were retroprosthetic membrane formation (40%), uveitis (20%), glaucoma (20%), tissue overgrowth (20%), early leakage (20%), late leakage (20%), aseptic necrosis (20%), retinal detachment (10%), endophthalmitis (10%), and granulation tissue growth (10%). CONCLUSIONS: Fyodorov-Zuev keratoprosthesis is an effective means of managing repeated corneal graft failure.

A hydrogel/particle composite with gradient in oxygen releasing microparticle for oxygenation of the cartilage-to-bone interface: Modeling and experimental viewpoints.

At the cartilage-to-bone interface, the residing cells are different with respects to metabolic requirements. Fabrication of a scaffold affording different metabolic needs of these cells can be taken account of a promoting step for regeneration of cartilage- to-bone interface. In the present study, a scaffold with a depth-dependent gradient of oxygen releasing microparticles was developed. To this end, oxygen releasing microparticles were fabricated from polylactic acid (PLA) and calcium peroxide and then dispersed in hydrogel precursor of functionalized pectin and fibroin. The microparticles were loaded in a hydrogel precursor solution in a gradient manner using a gradient mixing chamber. The mixing chamber was composed of two compartments filled with hydrogel precursors with different microparticle contents (10 and 30% w/w) and an interfacial mixing port. The velocity of microparticle loaded solution inside the gradient chamber was modeled using momentum balance Navier-Stokes equations. Moreover, spatial and temporal variations of microparticle concentration in the gradient chamber were modeled using mass transfer Navier-Stokes equations. Chemical, morphological and structural variations across the composite thickness were evaluated using microscopy and spectroscopy analyses. The model proposed by Navier-Stokes equation corroborated that the flow velocity was different in various domains of the mixing chamber and in the vicinity of the mixing port the velocity was substantially higher than the bulk flow. Moreover, the velocity profile showed gradual velocity changes from bulk to the mixing port. The model represented for microparticle concentration proved that microparticle content of precursor solution varied both spatially and temporally. As time goes by, the microparticle concentration gradually increased from about 10% w/w and approached to about 30% w/w at the end of the process. SEM micrographs from the cross-section of composite corroborated that microparticle density gradually increased from the lower to the upper surface. Spectroscopy confirmed that the oxygen releasing component, i.e. calcium peroxide, increased across the said direction. Oxygen measurement from successive sections of composite revealed that the amount of produced oxygen increased from the lowermost to the uppermost section. In conclusion, the hydrogel/particle composite with a gradient in oxygen releasing component can be a promising scaffold to satisfy the different metabolic needs of cells at the cartilage- to-bone interface. STATEMENT OF SIGNIFICANCE: Numerous tissue engineering scaffolds have been so far fabricated to recapitulate the gradient nature of cartilage- to-bone interface. Chemical, mechanical, structural and even electrical gradients of cartilage- to-bone interface have been tried to imitate by those scaffolds. However, scaffold with a gradient in metabolic features has not been developed yet. At the cartilage-to-bone interface, the residing cells are different with respects to metabolic requirements. Therefore, fabrication of a scaffold affording different metabolic needs of these cells can be taken account of a promoting step for regeneration of cartilage- to-bone interface. In the present study, a scaffold with a depth-dependent gradient of oxygen releasing microparticles was developed using a gradient making chamber. In addition to mathematical modeling of flow velocity and microparticle concentration in gradient making device, the depth-dependent changes in morphology, chemistry and structure of hydrogel/particle composite were experimentally evaluated.

Electroactive and antioxidant injectable in-situ forming hydrogels with tunable properties by polyethylenimine and polyaniline for nerve tissue engineering.

The injectable in-situ forming electroconductive hydrogels with antioxidant activity are promising candidates for nerve tissue engineering. In this study, we synthesized and developed a gelatin-graft-polyaniline/periodate-oxidized alginate hydrogel through the introduction of branched polyethylenimine (PEI) to improve the rheological properties. Moreover, antioxidant property, electroconductivity and the effect of external electrical stimulus on the nerve cell behavior were investigated. The results showed that by increasing the polyaniline content, the antioxidant activity, pore sizes, and swelling ratio of the hydrogel were increased, while the crosslinking density and storage modulus were decreased. The introduction of PEI accelerated the gelation time, decreased swelling ratio and pore size, and increased the storage modulus and crosslinking density. Cell studies showed that all formulations had supported the viability of P19 embryonic carcinoma cells with the neuritis elongation in the presence of the external electrical-stimulus. Gene expression of the neuronal markers, including Nestin, Pax-6, and ß-tubulin III, was increased in all hydrogels; In addition, electrical stimulation significantly elevated the expression of these markers in high polyaniline-content hydrogel compared to the polyaniline-free hydrogel. In conclusion, the results suggest that the prepared injectable electroconductive hydrogels can be a promising approach for neural tissue engineering.

A bifunctional electrospun nanocomposite wound dressing containing surfactin and curcumin: In vitro and in vivo studies.

A double-nozzle electrospinning technique was adopted in the present study to yield a novel bifunctional wound dressing composed of curcumin (Cur) and surfactin (Sur)-loaded poly(ε-caprolactone) (PCL)-gelatin (Gel). To comprehensively unveil the effect of both composition and drug molecules on the applicability, different dressings composed of PCL, Gel, and combination of the polymers with the drug molecules were fabricated. Besides the physicochemical properties, the in vitro and in vivo biological properties of prepared wound dressings were assessed. The results showed that increasing in the Cur from 0 to 3% (w/w) and Sur from 0 to 0.2 mg/mL caused a decrease in the elastic modulus on the one hand. On the other hand, the tensile strength and elongation at break experienced an increase in their values. The wettability, swelling capacity, and degradation rate of PCL improved significantly when both Gel and the drug molecules had been added. The dressings encompassing Sur (0.2 mg/mL) exhibited an excellent antibacterial activity after 24 h (>99%). Moreover, a sustained release of Cur up to 14 days was obtained. The in vitro cell compatibility tests implied a desirable result for all dressings without taking the composition into consideration. To complement the in vitro studies, the PCL/0.2Sur-Gel/3%Cur dressing was further assessed in vivo and the results revealed a significant improvement in the healing rate compared to control groups proofing its great potential for accelerated wound healing applications.

A hydrogel/particle composite with a gradient of oxygen releasing microparticle for concurrent osteogenic and chondrogenic differentiation in a single scaffold.

In the present study, a hydrogel/particle scaffold with a gradient of the oxygen releasing microparticles was developed. Hydrogel component was composed of the oxidized pectin and silk fibroin, whereas the microparticles were constituted from polylactic acid (PLA) and calcium peroxide (CPO). A controlled mixing of the suspensions with different content of the PLA/CPO microparticles conferred a gradient of microparticles in scaffold thickness in a manner that the microparticle content increased with moving from lower to upper face of the composite. Measurement of the scaffold mechanical properties corroborated that with moving from lower to upper face, the compressive modulus increased by 78 %. The measurement of the oxygen and calcium release from the successive sections of the composite revealed that the gradient of microparticle concentration resulted in the gradient of the released oxygen and calcium. MTT analysis proved that the gradient oxygen releasing composite did not induce any toxic effect on human adipose-derived mesenchymal stem cells (hAd-MSCs). Moreover, the cell culture on successive sections of the gradient composite confirmed that oxygen releasing composite substantially improved the cell viability and density comparing the pristine hydrogel and the non-oxygen releasing counterpart. The increase in microparticle content conferred a positive impact on the number of viable cells. The study of osteogenic (ALP, OCN and OPN) and chondrogenic (SOX9, AGG and COL ⅠⅠ) gene expression proved that the gradient composite parts with high microparticle content promoted osteogenesis, whereas the parts with low microparticle content encouraged chondrogenesis of mesenchymal stem cells.

Fabrication of oxygen and calcium releasing microcarriers with different internal structures for bone tissue engineering: Solid filled versus hollow microparticles.

The survival of cells in a three-dimensional scaffold until the ingrowth of blood vessels is an important challenge in bone tissue engineering. Oxygen generating biomaterials can provide the required oxygen and prevent hypoxia in a tissue-engineered scaffold. In this study, poly (L-lactic acid) (PLLA) microspheres loaded with synthesized calcium peroxide (CPO) nanoparticles were fabricated using two different methods, which resulted in hollow and solid filled internal structures. Catalase enzyme was grafted onto the microsphere surfaces to accelerate the conversion of hydrogen peroxide (H2O2) to oxygen and prevent the accumulation of H2O2 and cell damages. CPO loaded PLLA microspheres-graft-catalase could provide dissolved oxygen and calcium ions in release media up to 15 days. The oxygen release profile of solid filled microspheres was more sustained than the hollow structure, and the amount of calcium ions was higher for hollow microspheres due to the high loading content of CPO. MTT assay showed that CPO loaded PLLA microspheres without catalase exhibited a decrease in the cell viability below 75 %, and catalase grafting could prevent cytotoxicity. Human adipose-derived mesenchymal stem cells (hASCs) could adhere to the microsphere surfaces, maintain their morphology, and spread well. Based on these results, CPO loaded PLLA microspheres-graft-catalase, with the ability of cell carrying and controlled release of oxygen and calcium ions, can be a promising injectable cell microcarrier system for regeneration of bone tissue defects.

Oxygen-releasing nanofibers for breathable bone tissue engineering application.

Oxygen is a vital molecule for cell and tissue processes. Electrospun fibers have been extensively used as drug loading carriers due to possibility of well control over drug release with modulating fiber properties. However, they have not been used as depots for oxygen release. In the present study, an oxygen-releasing nanofibrous scaffold has been developed by electrospinning of polylactic acid/nano-calcium peroxide suspension with different polylactic acid concentrations (6.5 and 13% w/v). The electrospun fibers with calcium peroxide cargo provided oxygen content of 30-94 mmHg in a period of 14 days which lies well within the oxygen level of osseous tissue. The release profile of 13% polylactic acid fibers was different with that of 6.5% fibers with respects to the initial content of released oxygen and the release rate. Not only did 13% fibers supply oxygen with a slower rate, but also they resulted in a lower burst release of oxygen. Cell culture studies in hypoxia corroborated that 13% polylactic acid fibers better preserve cell viability comparing 6.5% counterparts as perceived by MTT assay. Moreover, they endowed more favored milieu for adherence, arrangement and migration of mesenchymal stem cells as confirmed by microscopy images. The oxygen-releasing fibers equally affected alkaline phosphatase, osteocalcin, and calcium deposition by mesenchymal stem cells most likely due to interplay between topographical and metabolic cues offered by 6.5 and 13% formulations.

Fabrication of amine-decorated nonspherical microparticles with calcium peroxide cargo for controlled release of oxygen.

Oxygen is an important signaling molecule which affects many behaviors of bone progenitor cells. Oxygen releasing biomaterials depend on their material and design are able to provide and modulate the desired oxygen for cells. To date, many oxygen releasing vehicles have been developed by incorporating microsized calcium peroxide (CPO) into polymeric matrixes. However, an oxygen releasing system based on nano CPO is still lacking. Not only can nanosized CPO provide more controllable oxygen release, but also can be loaded in vehicles of different shapes and sizes. Current research was conducted to take the advantages of nanomaterials as oxygen releasing components. To this end, CPO nanoparticles were synthesized using a hydrolysis-precipitation procedure and then loaded into the poly (lactide-co-glycolide) (PLGA) matrix via an electrospray process. The surface of PLGA/CaO2 particles was decorated with amine functionalities to render them more bioactive through a controlled aminolysis reaction. The studies on PLGA/CaO2 microparticles revealed that biconcave disk-like morphology with a mean diameter of 5.3 µm was formed. The particles persistently provide oxygen content of 35-67.5 mmHg up to 14 days which lies within the acceptable range for bone tissue engineering applications. PLGA/CaO2 microparticles induced 208 and 76% increase in number of viable mesenchymal cells on 6th and 14th days of cell seeding comparing PLGA counterparts. Furthermore, the expression of two bone biomarkers, that is, alkaline phosphatase and osteocalcin, at protein level as well as the extent of calcium deposition was increased in the presence of PLGA/CaO2 microparticles compared to PLGA ones.