Osteogenic potential of adipose stem cells on hydroxyapatite-functionalized decellularized amniotic membrane.

Amniotic membrane (AM) is an attractive source for bone tissue engineering because of its low immunogenicity, contains biomolecules and proteins, and osteogenic differentiation properties. Hydroxyapatite is widely used as bone scaffolds due to its biocompatibility and bioactivity properties. The aim of this study is to design and fabricate scaffold based on hydroxyapatite-coated decellularized amniotic membrane (DAM-HA) for bone tissue engineering purpose. So human amniotic membranes were collected from healthy donors and decellularized (DAM). Then a hydroxyapatite-coating was created by immersion in 10X SBF, under variable parameters of pH and incubation time. Hydroxyapatite-coating was characterized and the optimal sample was selected. Human adipose-derived mesenchymal stem cell behaviors were assessed on control, amniotic membrane, and coated amniotic membrane. The results of the SEM, MTT assay, and Live-Dead staining showed that DAM and DAM-HA support cell adhesion, viability and proliferation. Osteogenic differentiation was evaluated by assessment of alkaline phosphatase activity and expression of osteogenic markers. Maximum gene expression values compared to control occurred in 14 days for alkalin phosphatase, while the highest values for osteocalcin and osteopontin in 21 days. These gene expression values in DAM and DAM-HA for alkalin phosphatase is 6.41 and 8.47, for osteocalcin is 3.95 and 5.94 and for osteopontin is 5.59 and 9.9 respectively. The results of this study indicated DAM supports the survival and growth of stem cells. Also, addition of hydroxyapatite component to DAM promotes osteogenic differentiation while maintaining viability. Therefore, hydroxyapatite-coated decellularized amniotic membrane can be a promising choice 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.

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.

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.

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.

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.

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.

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.

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.

Development of an oxygen-releasing electroconductive in-situ crosslinkable hydrogel based on oxidized pectin and grafted gelatin for tissue engineering applications.

Injectable hydrogels with conductivity are highly desirable as scaffolds for the engineering of various electrical stimuli-responsive tissues, including nerve, muscle, retina, and bone. However, oxygen deprivation within scaffolds can lead to failure by causing cell necrosis. Therefore, an oxygen release conductive injectable hydrogel can serve as a promising support for the regeneration of such tissues. In the present study, H2O2-loaded polylactic acid microparticles were fabricated. Then, gelatin-graft-polypyrrole with various pyrrole contents and periodate-oxidized pectin were synthesized, and consequently, injectable conductive hydrogel/microparticle scaffolds, inside which catalase was grafted and trapped, were obtained. The results revealed that spherical particles with a mean diameter of 60.39 µm and encapsulation efficiency of 49.64 %, which persistently provided oxygen up to 14 days, were achieved. Investigations on hydrogels revealed that with the increase of pyrrole content of gelatin-graft-polypyrrole from 0 to 15 %, the swelling ratio, pore size, porosity, and conductivity were increased from 6.5 to 11.8, 173.13 µm-295.96 µm, 79.7%-93.8%, and from 0.06 mS/m to 2.14 mS/m, respectively. On the other hand, the crosslinking degree and compressive modulus of hydrogels were shown to decrease from 67.24%-27.35%, and from 214.1 kPa to 64.4 kPa, respectively. Moreover, all formulations supported cell viability and attachment. Overall, the hydrogel/particle scaffold with the merits of electrical conductivity, injectability, compatibility, and sustained oxygen release can be used as a tissue engineering scaffold, promoting the regeneration of electricity responsive tissues. Considering all the aforementioned characteristics and behavior of the fabricated scaffolds, they may be promising candidates for bone tissue engineering applications.