Nanoparticles for Targeted Drug Delivery to Cancer Stem Cells: A Review of Recent Advances.

Cancer stem cells (CSCs) are a subpopulation of cells that can initiate, self-renew, and sustain tumor growth. CSCs are responsible for tumor metastasis, recurrence, and drug resistance in cancer therapy. CSCs reside within a niche maintained by multiple unique factors in the microenvironment. These factors include hypoxia, excessive levels of angiogenesis, a change of mitochondrial activity from aerobic aspiration to aerobic glycolysis, an upregulated expression of CSC biomarkers and stem cell signaling, and an elevated synthesis of the cytochromes P450 family of enzymes responsible for drug clearance. Antibodies and ligands targeting the unique factors that maintain the niche are utilized for the delivery of anticancer therapeutics to CSCs. In this regard, nanomaterials, specifically nanoparticles (NPs), are extremely useful as carriers for the delivery of anticancer agents to CSCs. This review covers the biology of CSCs and advances in the design and synthesis of NPs as a carrier in targeting cancer drugs to the CSC subpopulation of cancer cells. This review includes the development of synthetic and natural polymeric NPs, lipid NPs, inorganic NPs, self-assembling protein NPs, antibody-drug conjugates, and extracellular nanovesicles for CSC targeting.

Thermoresponsive Nanogels Based on Different Polymeric Moieties for Biomedical Applications.

Nanogels, or nanostructured hydrogels, are one of the most interesting materials in biomedical engineering. Nanogels are widely used in medical applications, such as in cancer therapy, targeted delivery of proteins, genes and DNAs, and scaffolds in tissue regeneration. One salient feature of nanogels is their tunable responsiveness to external stimuli. In this review, thermosensitive nanogels are discussed, with a focus on moieties in their chemical structure which are responsible for thermosensitivity. These thermosensitive moieties can be classified into four groups, namely, polymers bearing amide groups, ether groups, vinyl ether groups and hydrophilic polymers bearing hydrophobic groups. These novel thermoresponsive nanogels provide effective drug delivery systems and tissue regeneration constructs for treating patients in many clinical applications, such as targeted, sustained and controlled release.

Free and hydrogel encapsulated exosome-based therapies in regenerative medicine.

Over the last few decades, mesenchymal stem cells-derived exosomes (MSCs-Ex) have attracted a lot of attention as a therapeutic tool in regenerative medicine. Exosomes are extracellular vehicles (EVs) that play important roles in cell-cell communication through various processes such as stress response, senescence, angiogenesis, and cell differentiation. Success in the field of regenerative medicine sparked exploration of the potential use of exosomes as key therapeutic effectors of MSCs to promote tissue regeneration. Various approaches including direct injection, intravenous injection, intraperitoneal injection, oral administration, and hydrogel-based encapsulation have been exploited to deliver exosomes to target tissues in different disease models. Despite significant advances in exosome therapy, it is unclear which approach is more effective for administering exosomes. Herein, we critically review the emerging progress in the applications of exosomes in the form of free or association with hydrogels as therapeutic agents for applications in regenerative medicine.

Material properties and cell compatibility of poly(γ-glutamic acid)-keratin hydrogels.

Given the great demand for biopolymer and protein-based products from renewable resources, synthesis of a keratin-based hydrogel is presented herein. In this work, a novel hydrogel of poly(γ-glutamic acid) (γ-PGA) and keratin was synthesized through facile EDC·HCl/HOBt chemistry. Since keratin main chain is rich in amino side groups, carboxyl groups in γ-PGA were crosslinked with multi terminated amine groups in keratin. In the following, the hydrogel characteristics, including swelling ratio (2010% at molar ratio of HOBt/EDC = 0.105), in vitro degradation and mass loss (about 20% at day 21 for the aforementioned sample), chemical decomposition and the rheological properties were investigated. The chemical activator agents, enhanced the elastic modulus of swollen hydrogel from around 1000 to 4000 Pa by increasing the crosslinking degree. Despite good biocompatibility for cell growth, some kind of self-assembled keratin hydrogels are not suitable for microscopic observation while the γ-PGA-Keratin hydrogel in our study is transparent. The γ-PGA-Keratin hydrogels possess significant features of rapid hydrogel formation in seconds, maximum swelling ratio of about 2500% maximum elastic modulus (stiffness) of about 4.5 kPa (for the swollen sample) with controllable matrix pore size. For further application, the biocompatibility of the γ-PGA-Keratin hydrogel was assessed by live/dead assay. Recent studies have demonstrated the effect of hydrogel porosity, water absorbing and stiffness on cell spreading, proliferation and differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells could be differentiated into various cell fates depending on the elastic modulus of materials they are cultured on. We carried out a statistical study (to skip the cell work labor) to predetermine the proper working span in which we can gain a hydrogel to cover all features needed to be applied for some application like cartilage repair.

Plasmin-Cleavable Nanoparticles for On-Demand Release of Morphogens in Vascularized Osteogenesis.

The objective of this work was to engineer self-assembled nanoparticles (NPs) for on-demand release of bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) in response to enzymes secreted by the migrating human mesenchymal stem cells (hMSCs) and human endothelial colony forming cells (ECFCs) to induce osteogenesis and vasculogenesis. Gene expression profiling experiments revealed that hMSCs and ECFCs, encapsulated in osteogenic/vasculogenic hydrogels, expressed considerable levels of plasminogen, urokinase plasminogen activator and its receptor uPAR, and tissue plasminogen activator. Therefore, the plasmin-cleavable lysine-phenylalanine-lysine-threonine (KFKT) was used to generate enzymatically cleavable NPs. The acetyl-terminated, self-assembling peptide glycine-(phenylalanine)3GFFF-ac and the plasmin-cleavable GGKFKTGG were reacted with the cysteine-terminated CGGK(Fmoc/MTT) peptide through the MTT and Fmoc termini, respectively. The difunctional peptide was conjugated to polyethylene glycol diacrylate (PEGDA) with molecular weights (MW) ranging from 0.5 to 7.5 kDa, and the chain ends of the PEG-peptide conjugate were terminated with succinimide groups. After self-assembly in aqueous solution, BMP2 was grafted to the self-assembled, plasmin-cleavable PEG-based (PxSPCP) NPs for on-demand release. The NPs' stability in aqueous solution and that of the grafted BMP2 were strongly dependent on PEG MW. P2SPCP NPs showed high particle size stability, BMP2 grafting efficiency, grafted protein stability, and high extent of osteogenic differentiation of hMSCs. The localized and on-demand release of BMP2 from PxSPCP NPs coencapsulated with hMSCs in the linear polyethylene glycol-co-lactide acrylate patterned hydrogel with microchannels encapsulating hMSCs + ECFCs and VEGF-conjugated nanogels resulted in the highest extent of osteogenic and vasculogenic differentiation of the encapsulated cells compared to directly added BMP2/VEGF. The on-demand release of BMP2 from PxSPCP NPs not only enhances osteogenesis and vasculogenesis but also potentially reduces many undesired side effects of BMP2 therapy in bone regeneration.

Regenerative Scar-Free Skin Wound Healing.

IMPACT STATEMENT: Millions of people every year develop scars in response to skin injuries after surgery, trauma, or burns with significant undesired physical and psychological effects. This review provides an update on engineering strategies for scar-free wound healing and discusses the role of different cell types, growth factors, cytokines, and extracellular components in regenerative wound healing. The use of pro-regenerative matrices combined with engineered cells with less intrinsic potential for fibrogenesis is a promising strategy for achieving scar-free skin tissue regeneration.

Microwave-assisted and one-step synthesis of PEG passivated fluorescent carbon dots from gelatin as an efficient nanocarrier for methotrexate delivery.

A green and simple process for preparing the polyethylene glycol passivated fluorescent carbon dots (CDs-PEG) have been studied by a microwave pyrolysis method, using gelatin and PEG as starting materials. This method is very effective for development of carbon-based quantum dots from gelatin with high quantum yield (QY). The synthesized CDs-PEG were found to emit blue photoluminescence (PL) with a maximum QY of 34%. At the following research, we investigated the effect of the presence of PEG on PL intensity, and the result showed that CDs-PEG becomes stronger PL properties than pure CDs from gelatin. The synthesized CDs-PEG were characterized by FTIR, TEM, UV-vis, PL, zeta potential and XRD analyses. The anticancer performance of developed CDs-PEG was evaluated by in vitro tests such as MTT assay and fluorescence microscopy analyses. The examination of CDs-PEG as an anti-cancer drug nanocarrier for methotrexate (MTX) illustrated a better antitumor efficacy than free MTX due to its enhanced nuclear delivery in vitro, which resulting in highly effective tumour growth inhibition and improving targeted cancer therapy in clinical medicine.

Sequential Zonal Chondrogenic Differentiation of Mesenchymal Stem Cells in Cartilage Matrices.

IMPACT STATEMENT: The higher regenerative capacity of fetal articular cartilage compared with the adult is rooted in differences in cell density and matrix composition. We hypothesized that the zonal organization of articular cartilage can be engineered by encapsulation of mesenchymal stem cells in a single superficial zone-like matrix followed by sequential addition of zone-specific growth factors within the matrix, similar to the process of fetal cartilage development. The results demonstrate that the zonal organization of articular cartilage can potentially be regenerated using an injectable, monolayer cell-laden hydrogel with sequential release of growth factors.

Fabrication of in situ polymerized poly(butylene succinate-co-ethylene terephthalate)/hydroxyapatite nanocomposite to fibrous scaffolds for enhancement of osteogenesis.

A combination of elastic poly(butylene succinate-co-ethylene terephthalate) and rigid nano-hydroxyapatite were used to prepare an in-situ synthesized nanocomposite mimicing bone structure. The microstructure, morphology, and dispersion of nanoparticles in the nanocomposites were studied using proton nuclear magnetic resonance (1 HNMR), scanning electron microscope (SEM), and transmission electron microscopy (TEM), respectively. Then, electrospinning method was used to produce nanofiber matrix with lowest fiber diameter. Presence of chemical bonds among nanoparticles and polymer leads to the excellent particle dispersion and solution phase stability. SEM results show that continuous and bead-free nanofibers were produced and incorporating nanoparticle slightly increased fibers diameter. Elastic modulus, tensile strength, crystallinity, hydrophilicity and hydrolytic degradability of resulted nanofiber increased with nanoparticle but elongation at break slightly decreased. Proliferation and osteogenic differentiation of human mesenchymal stem cell significantly improved by introducing nanoparticle which indicate that electrospun nanofibers could be used as scaffolds for bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2622-2631, 2017.

3D Cell Culture in Micropatterned Hydrogels Prepared by Photomask, Microneedle, or Soft Lithography Techniques.

Despite the advantages of three-dimensional (3D) hydrogels for cell culture over traditional 2D plates, their clinical application is limited by inability to recapitulate the micro-architecture of complex tissues. Micropatterning can be employed to modify the homogenous micro-architecture of hydrogels. Three techniques for cell encapsulation in 3D micropatterned gels are described. The photomask and micromold techniques are used for cell encapsulation in relatively shallow patterns like disks or short rectangles but due to the presence of PDMS mold, the resolution of micromold technique is potentially higher than the photomask. The microneedle technique is often used for cell encapsulation in relatively deep microchannels within any geometry.

Covalently immobilized VEGF-mimicking peptide with gelatin methacrylate enhances microvascularization of endothelial cells.

Clinically usable tissue-engineered constructs are currently limited due to their inability of forming microvascular networks necessary for adequate cellular oxygen and nutrient supply upon implantation. The aim of this study is to investigate the conditions necessary for microvascularization in a tissue-engineered construct using vascular endothelial growth factor (VEGF). The construct was made of gelatin methacrylate (GelMA) based cell-laden hydrogel system, which was then covalently linked with VEGF-mimicking peptide (AcQK), using human umbilical vein endothelial cells (HUVECs) as the model cell. The results of the mechanics and gene expression analysis indicated significant changes in mechanical properties and upregulation of vascular-specific genes. The major finding of this study is that the increased expression of vascular-specific genes could be achieved by employing AcQK in the GelMA based hydrogel system, leading to accelerated microvascularization. We conclude that GelMA with covalently-linked angiogenic peptide is a useful tissue engineered construct suitable for microvascularization. STATEMENT OF SIGNIFICANCE: (1) This study reports the conditions necessary for microvascularization in a tissue-engineered construct using vascular endothelial growth factor (VEGF). (2) The construct was made of gelatin methacrylate based cell-laden hydrogel system. (3) There is a significant change observed in mechanical properties and upregulation of vascular-specific genes, in particular CD34, when AcQK is used. (4) The major finding of this study is that the increased expression of vascular-specific genes, i.e., CD34 could be achieved by employing AcQK in the GelMA based hydrogel system, leading to accelerated microvascularization.

Synthesis and Characterization of Photo-Cross-Linkable Keratin Hydrogels for Stem Cell Encapsulation.

The objective of this work was to synthesize an injectable and photopolymerizable hydrogel based on keratin extracted from poultry feather for encapsulation and delivery of stem cells in tissue regeneration. Since feather keratin is rich in cysteine residue, allylation of sulfhydryl groups was used for functionalization of keratin. Keratin was extracted from feather barbs by reducing the disulfide bonds in cysteine residues to sulfhydryl groups (-SH). Next, the free thiol groups were converted to dehydroalanine (Dha) by oxidative elimination using O-(2,4,6-trimethylbenzenesulfonyl) hydroxylamine. Then, the Dha moieties were converted to s-allyl cysteine by reaction with allyl mercaptan to produce keratin allyl thioether (KeratATE) biopolymer. Human mesenchymal stem cell (hMSCs) were suspended in the aqueous solution of KeratATE, injected into a mold, and photopolymerized to generate a KeratATE hydrogel encapsulating hMSCs. The freeze-dried photo-cross-linked KeratATE hydrogels had a porous, interconnected, honeycomb microstructure with pore sizes in the 20-60 µm range. The compressive modulus of the hydrogels ranged from 1 to 8 kPa depending on KeratATE concentration. KeratATE hydrogels had <5% mass loss in collagenase solution after 21 days of incubation, whereas the mass loss was 15% in trypsin solution. Degradation of KeratATE hydrogel was strongly dependent on trypsin concentration but independent of collagenase. hMSCs proliferated and adopted an elongated spindle-shape morphology after seeding on KeratATE hydrogel. KeratATE hydrogel supported differentiation of the encapsulated hMSCs to the osteogenic and chondrogenic lineages to the same extent as those hMSCs encapsulated in gelatin methacryloyl hydrogel. The results suggest that keratin allyl thioether hydrogel with controllable degradation is a viable matrix for encapsulation and delivery of stem cells in tissue regeneration.

Comparative effect of physicomechanical and biomolecular cues on zone-specific chondrogenic differentiation of mesenchymal stem cells.

Current tissue engineering approaches to regeneration of articular cartilage rarely restore the tissue to its normal state because the generated tissue lacks the intricate zonal organization of the native cartilage. Zonal regeneration of articular cartilage is hampered by the lack of knowledge for the relation between physical, mechanical, and biomolecular cues and zone-specific chondrogenic differentiation of progenitor cells. This work investigated in 3D the effect of TGF-ß1, zone-specific growth factors, optimum matrix stiffness, and adding nanofibers on the expression of chondrogenic markers specific to the superficial, middle, and calcified zones of articular cartilage by the differentiating human mesenchymal stem cells (hMSCs). Growth factors included BMP-7, IGF-1, and hydroxyapatite (HA) for the superficial, middle, and calcified zones, respectively; optimum matrix stiffness was 80 kPa, 2.1 MPa, and 320 MPa; and nanofibers were aligned horizontal, random, and perpendicular to the gel surface. hMSCs with zone-specific cell densities were encapsulated in engineered hydrogels and cultured with or without TGF-ß1, zone-specific growth factor, optimum matrix modulus, and fiber addition and cultured in basic chondrogenic medium. The expression of encapsulated cells was measured by mRNA, protein, and biochemical analysis. Results indicated that zone-specific matrix stiffness had a dominating effect on chondrogenic differentiation of hMSCs to the superficial and calcified zone phenotypes. Addition of aligned nanofibers parallel to the direction of gel surface significantly enhanced expression of Col II in the superficial zone chondrogenic differentiation of hMSCs. Conversely, biomolecular factor IGF-1 in combination with TGF-ß1 had a dominating effect on the middle zone chondrogenic differentiation of hMSCs. Results of this work could potentially lead to the development of multilayer grafts mimicking the zonal organization of articular cartilage.

Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel.

Reconstruction of large bone defects is limited by insufficient vascularization and slow bone regeneration. The objective of this work was to investigate the effect of spatial and temporal release of recombinant human bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) on the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) encapsulated in a patterned hydrogel. Nanogels (NGs) based on polyethylene glycol (PEG) macromers chain-extended with short lactide (L) and glycolide (G) segments were used for grafting and timed-release of BMP2 and VEGF. NGs with 12kDa PEG molecular weight (MW), 24 LG segment length, and 60/40L/G ratio (P12-II, NG(10)) released the grafted VEGF in 10days. NGs with 8kDa PEG MW, 26 LG segment length, and 60/40L/G ratio (P8-I, NG(21)) released the grafted BMP2 in 21days. hMSCs and NG-BMP2 were encapsulated in a patterned matrix based on acrylate-functionalized lactide-chain-extended star polyethylene glycol (SPELA) hydrogel and microchannel patterns filled with a suspension of hMSCs+ECFCs and NG-VEGF in a crosslinked gelatin methacryloyl (GelMA) hydrogel. Groups included patterned constructs without BMP2/VEGF (None), with directly added BMP2/VEGF, and NG-BMP2/NG-VEGF. Based on the results, timed-release of VEGF in the microchannels in 10days from NG(10) and BMP2 in the matrix in 21days from NG(21) resulted in highest extent of osteogenic and vasculogenic differentiation of the encapsulated hMSCs and ECFCs compared to direct addition of VEGF and BMP2. Further, timed-release of VEGF from NG(10) in hMSC+ECFC encapsulating microchannels and BMP2 from NG(21) in hMSC encapsulating matrix sharply increased bFGF expression in the patterned constructs. The results suggest that mineralization and vascularization are coupled by localized secretion of paracrine signaling factors by the differentiating hMSCs and ECFCs.

Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization.

Morphogenic Peptides in Regeneration of Load Bearing Tissues.

Morphogenic proteins due to their short half-life require high doses of growth factors in regeneration of load bearing tissues which leads to undesirable side effects. These side effects include bone overgrowth, tumor formation and immune reaction. An alternative approach to reduce undesirable side effects of proteins in regenerative medicine is to use morphogenic peptides derived from the active domains of morphogenic proteins or soluble and insoluble components of the extracellular matrix of mineralized load bearing tissues to induce differentiation of progenitor cells, mineralization, maturation and bone formation. In that regard, many peptides with osteogenic activity have been discovered. These include peptides derived from bone morphogenic proteins (BMPs), those based on interaction with integrin and heparin-binding receptors, collagen derived peptides, peptides derived from other soluble ECM proteins such as bone sialoprotein and enamel matrix proteins, and those peptides derived from vasculoinductive and neuro-inductive proteins. Although these peptides show significant osteogenic activity in vitro and increase mineralization and bone formation in animal models, they are not widely used in clinical orthopedic applications as an alternative to morphogenic proteins. This is partly due to the limited availability of data on structure and function of morphogenic peptides in physiological medium, particularly in tissue engineered scaffolds. Due to their amphiphilic nature, peptides spontaneously self-assemble and aggregate into micellar structures in physiological medium. Aggregation alters the sequence of amino acids in morphogenic peptides that interact with cell surface receptors thus affecting osteogenic activity of the peptide. Aggregation and micelle formation can dramatically reduce the active concentration of morphogenic peptides with many-fold increase in peptide concentration in physiological medium. Other factors that affect bioactivity are the non-specific interaction of morphogenic peptides with lipid bilayer of the cell membrane, interaction of the peptide with cell surface receptors that do not specifically induce osteogenesis leading to less-than-optimal osteogenic activity of the peptide, and less-than-optimal interaction of the peptide with osteogenic receptors on the cell surface. Covalent attachment or physical interaction with the tissue engineered matrix can also alter the bioactivity of morphogenic peptides and lead to a lower extent of osteogenesis and bone formation. This chapter reviews advances in discovery of morphogenic peptide, their structural characterization, and challenges in using morphogenic peptides in clinical applications as growth factors in tissue engineered devices for regeneration of load bearing tissues.

Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells.

INTRODUCTION: The growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells' tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment. METHODS: Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers. RESULTS: The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 µm. CONCLUSION: The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells' tissue origin.

Osteogenic differentiation of human mesenchymal stem cells in freeze-gelled chitosan/nano ß-tricalcium phosphate porous scaffolds crosslinked with genipin.

The objective of this work was to investigate material properties and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in genipin (GN) crosslinked chitosan/nano ß-tricalcium phosphate (CS/nano ß-TCP) scaffolds, and compare the results with tripolyphosphate (TPP) crosslinked scaffolds. Porous crosslinked CS/nano ß-TCP scaffolds were produced by freeze-gelation using GN (CBG scaffold) and TPP (CBT scaffold) as crosslinkers. The prepared CBT and CBG scaffolds were characterized with respect to porosity, pore size, water content, wettability, compressive strength, mass loss, and osteogenic differentiation of hMSCs. All scaffolds displayed interconnected honeycomb-like microstructures. There was a significant difference between the average pore size, porosity, contact angle, and percent swelling of CBT and CBG scaffolds. The average pore size of CBG scaffolds was higher than CBT, the porosity of CBG was lower than CBT, the water contact angle of CBG was higher than CBT, and the percent swelling of CBG was lower than CBT. At a given crosslinker concentration, there was not a significant difference in compressive modulus and mass loss of CBG and CBT scaffolds. Metabolic activity of hMSCs seeded in CBG scaffolds was slightly higher than CBT. Furthermore, CBG scaffolds displayed slightly higher extent of mineralization after 21 days of incubation in osteogenic medium compared to CBT.

Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs.

A developmentally inspired combined mechanical and biochemical signaling approach on zonal lineage commitment of mesenchymal stem cells in articular cartilage regeneration.

Articular cartilage is organized into multiple zones including superficial, middle and calcified zones with distinct cellular and extracellular components to impart lubrication, compressive strength, and rigidity for load transmission to bone, respectively. During native cartilage tissue development, changes in biochemical, mechanical, and cellular factors direct the formation of stratified structure of articular cartilage. The objective of this work was to investigate the effect of combined gradients in cell density, matrix stiffness, and zone-specific growth factors on the zonal organization of articular cartilage. Human mesenchymal stem cells (hMSCs) were encapsulated in acrylate-functionalized lactide-chain-extended polyethylene glycol (SPELA) gels simulating cell density and stiffness of the superficial, middle and calcified zones. The cell-encapsulated gels were cultivated in a medium supplemented with growth factors specific to each zone and the expression of zone-specific markers was measured with incubation time. Encapsulation of 60 × 10(6) cells per mL hMSCs in a soft gel (80 kPa modulus) and cultivation with a combination of TGF-ß1 (3 ng mL(-1)) and BMP-7 (100 ng mL(-1)) led to the expression of markers for the superficial zone. Conversely, encapsulation of 15 × 10(6) cells per mL hMSCs in a stiff gel (320 MPa modulus) and cultivation with a combination of TGF-ß1 (30 ng mL(-1)) and hydroxyapatite (3%) led to the expression of markers for the calcified zone. Further, encapsulation of 20 × 10(6) cells per mL hMSCs in a gel with 2.1 MPa modulus and cultivation with a combination of TGF-ß1 (30 ng mL(-1)) and IGF-1 (100 ng mL(-1)) led to up-regulation of the middle zone markers. Results demonstrate that a developmental approach with gradients in cell density, matrix stiffness, and zone-specific growth factors can potentially regenerate zonal structure of the articular cartilage.