Toward 3D Bioprinting of Osseous Tissue of Predefined Shape Using Single-Matrix Cell-Bioink Constructs.

Engineering living bone tissue of defined shape on-demand has remained a challenge. 3D bioprinting (3DBP), a biofabrication process capable of yielding cell constructs of defined shape, when combined with developmental engineering can provide a possible path forward. Through the development of a bioink possessing appropriate rheological properties to carry a high cell load and concurrently yield physically stable structures, printing of stable, cell-laden, single-matrix constructs of anatomical shapes is realized without the need for fugitive or support phases. Using this bioink system, constructs of hypertrophic cartilage of predesigned geometry are engineered in vitro by printing human mesenchymal stromal cells at a high density to drive spontaneous condensation and implanted in nude mice to evoke endochondral ossification. The implanted constructs retain their prescribed shape over a 12-week period and undergo remodeling to yield ossicles of the designed shape with neovascularization. Microcomputed tomography, histological, and immunohistochemistry assessments confirm bone tissue characteristics and the presence of human cells. These results demonstrate the potential of 3DBP to fabricate complex bone tissue for clinical application.

Mesenchymal-endothelial nexus in breast cancer spheroids induces vasculogenesis and local invasion in a CAM model.

Interplay between non-cancerous cells (immune, fibroblasts, mesenchymal stromal cells (MSC), and endothelial cells (EC)) has been identified as vital in driving tumor progression. As studying such interactions in vivo is challenging, ex vivo systems that can recapitulate in vivo scenarios can aid in unraveling the factors impacting tumorigenesis and metastasis. Using the synthetic tumor microenvironment mimics (STEMs)-a spheroid system composed of breast cancer cells (BCC) with defined human MSC and EC fractions, here we show that EC organization into vascular structures is BC phenotype dependent, and independent of ERα expression in epithelial cancer cells, and involves MSC-mediated Notch1 signaling. In a 3D-bioprinted model system to mimic local invasion, MDA STEMs collectively respond to serum gradient and form invading cell clusters. STEMs grown on chick chorioallantoic membrane undergo local invasion to form CAM tumors that can anastomose with host vasculature and bear the typical hallmarks of human BC and this process requires both EC and MSC. This study provides a framework for developing well-defined in vitro systems, including patient-derived xenografts that recapitulate in vivo events, to investigate heterotypic cell interactions in tumors, to identify factors promoting tumor metastasis-related events, and possibly drug screening in the context of personalized medicine.

Double-interpenetrating nanostructured networks of marine polysaccharides possessing properties comparable to synthetic polymers.

Sustainable circular economy requires materials that possess a property profile comparable to synthetic polymers and, additionally, processing and sourcing of raw materials that have a small environmental footprint. Here, we present a paradigm for processing marine biopolymers into materials that possess both elastic and plastic behavior within a single system involving a double-interpenetrating polymer network comprising the elastic phase of dynamic physical cross-links and stress-dissipating ionically cross-linked domains. As a proof of principle, films possessing more than twofold higher elastic modulus, ultimate tensile strength, and yield stress than those of polylactic acid were realized by blending two water-soluble marine polysaccharides, namely alginic acid (Alg) with physically cross-linkable carboxylated agarose (CA) followed by ionic cross-linking with a divalent cation. Dried CAAlg films showed homogeneous nano-micro-scale domains, with yield stress and size of the domains scaling inversely with calcium concentration. Through surface activation/cross-linking using calcium, CAAlg films could be further processed using wet bonding to yield laminated structures with interfacial failure loads (13.2 ± 0.81 N) similar to the ultimate loads of unlaminated films (10.09 ± 1.47 N). Toward the engineering of wood-marine biopolymer composites, an array of lines of CAAlg were printed on wood veneers (panels), dried, and then bonded following activation with calcium to yield fully bonded wood two-ply laminate. The system presented herein provides a blueprint for the adoption of marine algae-derived polysaccharides in the development of sustainable high-performance materials.

3D Printed Solutions for Spheroid Engineering and Cancer Research.

In multicellular organisms, cells are organized in a 3-dimensional framework and this is essential for organogenesis and tissue morphogenesis. Systems to recapitulate 3D cell growth are therefore vital for understanding development and cancer biology. Cells organized in 3D environments can evolve certain phenotypic traits valuable to physiologically relevant models that cannot be accessed in 2D culture. Cellular spheroids constitute an important aspect of in vitro tumor biology and they are usually prepared using the hanging drop method. Here a 3D printed approach is demonstrated to fabricate bespoke hanging drop devices for the culture of tumor cells. The design attributes of the hanging drop device take into account the need for high-throughput, high efficacy in spheroid formation, and automation. Specifically, in this study, custom-fit, modularized hanging drop devices comprising of inserts (Q-serts) were designed and fabricated using fused filament deposition (FFD). The utility of the Q-serts in the engineering of unicellular and multicellular spheroids-synthetic tumor microenvironment mimics (STEMs)-was established using human (cancer) cells. The culture of spheroids was automated using a pipetting robot and bioprinted using a custom bioink based on carboxylated agarose to simulate a tumor microenvironment (TME). The spheroids were characterized using light microscopy and histology. They showed good morphological and structural integrity and had high viability throughout the entire workflow. The systems and workflow presented here represent a user-focused 3D printing-driven spheroid culture platform which can be reliably reproduced in any research environment and scaled to- and on-demand. The standardization of spheroid preparation, handling, and culture should eliminate user-dependent variables, and have a positive impact on translational research to enable direct comparison of scientific findings.

Reversible, ß-sheet-dependent self-assembly of the phosphoprotein phosvitin is controlled by the concentration and valency of cations.

The hyperphosphorylated protein phosvitin (PV) undergoes a pH-dependent transition between PII- and ß-sheet secondary structures, a process deemed crucial for its role in the promotion of biogenic apatite formation. The transition occurs surprisingly slowly (minutes to hours). This is consistent with a slow aggregation process involving ionic interactions of charged groups on the protein surface. Herein, we determined the associated transition pK values and time constants through matrix least-squares (MLS) global fitting of a series of pH- and time-dependent circular dichroism (CD) spectra recorded in the presence of different mono-, bi- and trivalent cations. Supporting our results with dynamic light scattering data, we clearly identified a close correlation of ß-sheet transition and the formation of small aggregates at low pH. This process is inhibited in the presence of all tested cations with the strongest effects for trivalent cations (Fe3+ and Al3+). In the presence of Ca2+ and Mg2+, larger higher-order particles are formed from PV in the ß-sheet conformation, as identified from the interpretation of differential scattering observed in the CD spectra. Our observations are consistent with the existence of a multi-step equilibrium between aggregated and non-aggregated species of PV. The equilibrium is highly sensitive to the environment pH and salt concentration with exceptional behavior in the presence of divalent cations such as Ca2+ and Mg2+.

Biobridge: An Outlook on Translational Bioinks for 3D Bioprinting.

3D-bioprinting (3DBP) possesses several elements necessary to overcome the deficiencies of conventional tissue engineering, such as defining tissue shape a priori, and serves as a bridge to clinical translation. This transformative potential of 3DBP hinges on the development of the next generation of bioinks that possess attributes for clinical use. Toward this end, in addition to physicochemical characteristics essential for printing, bioinks need to possess proregenerative attributes, while enabling printing of stable structures with a defined biological function that survives implantation and evolves in vivo into functional tissue. With a focus on bioinks for extrusion-based bioprinting, this perspective review advocates a rigorous biology-based approach to engineering bioinks, emphasizing efficiency, reproducibility, and a streamlined translation process that places the clinical endpoint front and center. A blueprint for engineering the next generation of bioinks that satisfy the aforementioned performance criteria for various translational levels (TRL1-5) and a characterization tool kit is presented.

Extrusion-Based 3D Bioprinting of Gradients of Stiffness, Cell Density, and Immobilized Peptide Using Thermogelling Hydrogels.

To study biological processes in vitro, biomaterials-based engineering solutions to reproduce the gradients observed in tissues are necessary. We present a platform for the 3D bioprinting of functionally graded biomaterials based on carboxylated agarose, a bioink amendable by extrusion bioprinting. Using this bioink, objects with a gradient of stiffness and gradient of cell concentration were printed. Functionalization of carboxylated agarose with maleimide moieties that react in minutes with a cysteine-terminated cell-adhesion peptide allowed us to print objects with a gradient of an immobilized peptide. This approach paves the way toward the development of tissue mimics with gradients.

Hydrogel-Forming Algae Polysaccharides: From Seaweed to Biomedical Applications.

With the increasing growth of the algae industry and the development of algae biorefinery, there is a growing need for high-value applications of algae-extracted biopolymers. The utilization of such biopolymers in the biomedical field can be considered as one of the most attractive applications but is challenging to implement. Historically, polysaccharides extracted from seaweed have been used for a long time in biomedical research, for example, agarose gels for electrophoresis and bacterial culture. To overcome the current challenges in polysaccharides and help further the development of high-added-value applications, an overview of the entire polysaccharide journey from seaweed to biomedical applications is needed. This encompasses algae culture, extraction, chemistry, characterization, processing, and an understanding of the interactions of soft matter with living organisms. In this review, we present algae polysaccharides that intrinsically form hydrogels: alginate, carrageenan, ulvan, starch, agarose, porphyran, and (nano)cellulose and classify these by their gelation mechanisms. The focus of this review further lays on the culture and extraction strategies to obtain pure polysaccharides, their structure-properties relationships, the current advances in chemical backbone modifications, and how these modifications can be used to tune the polysaccharide properties. The available techniques to characterize each organization scale of a polysaccharide hydrogel are presented, and the impact on their interactions with biological systems is discussed. Finally, a perspective of the anticipated development of the whole field and how the further utilization of hydrogel-forming polysaccharides extracted from algae can revolutionize the current algae industry are suggested.

Advanced Bioink for 3D Bioprinting of Complex Free-Standing Structures with High Stiffness.

One of the challenges in 3D-bioprinting is the realization of complex, volumetrically defined structures, that are also anatomically accurate and relevant. Towards this end, in this study we report the development and validation of a carboxylated agarose (CA)-based bioink that is amenable to 3D printing of free-standing structures with high stiffness at physiological temperature using microextrusion printing without the need for a fugitive phase or post-processing or support material (FRESH). By blending CA with negligible amounts of native agarose (NA) a bioink formulation (CANA) which is suitable for printing with nozzles of varying internal diameters under ideal pneumatic pressure was developed. The ability of the CANA ink to exhibit reproducible sol-gel transition at physiological temperature of 37 °C was established through rigorous characterization of the thermal behavior, and rheological properties. Using a customized bioprinter equipped with temperature-controlled nozzle and print bed, high-aspect ratio objects possessing anatomically-relevant curvature and architecture have been printed with high print reproducibility and dimension fidelity. Objects printed with CANA bioink were found to be structurally stable over a wide temperature range of 4 °C to 37 °C, and exhibited robust layer-to-layer bonding and integration, with evenly stratified structures, and a porous interior that is conducive to fluid transport. This exceptional layer-to-layer fusion (bonding) afforded by the CANA bioink during the print obviated the need for post-processing to stabilize printed structures. As a result, this novel CANA bioink is capable of yielding large (5-10 mm tall) free-standing objects ranging from simple tall cylinders, hemispheres, bifurcated 'Y'-shaped and 'S'-shaped hollow tubes, and cylinders with compartments without the need for support and/or a fugitive phase. Studies with human nasal chondrocytes showed that the CANA bioink is amenable to the incorporation of high density of cells (30 million/mL) without impact on printability. Furthermore, printed cells showed high viability and underwent mitosis which is necessary for promoting remodeling processes. The ability to print complex structures with high cell densities, combined with excellent cell and tissue biocompatibility of CA bodes well for the exploitation of CANA bioinks as a versatile 3D-bioprinting platform for the clinical translation of regenerative paradigms.

Unraveling the role of ß1 integrin isoforms in cRGD-mediated uptake of nanoparticles bearing hydrophilized alkyne moieties in epithelial and endothelial cells.

The uptake and trafficking of NPs is impacted by several attributes such as size, shape, surface charge and importantly by surface ligands that can interact with the cell plasma membrane. We envision that NPs which can be readily modified in aqueous environments will be key to engineering patient-specific nanotherapeutics. Towards such systems that can be functionalized "on demand" in aqueous environments, an α-ω epoxy ester monomer that bears an alkyne group at the end of an oligoethylene glycol moiety was designed and synthesized. Copolymerization of this monomer with ε-caprolactone yielded polymers that present hydrophilized alkyne groups along the backbone. This enabled the direct modification of the surface of NPs, as suspensions in aqueous phase, with cell interaction peptides such cyclic-arginine-glycine-aspartic acid (cRGD) using the "click reaction". Uptake of cRGD modified NPs (cRGD-NPs) in human endothelial and tumor epithelial cells revealed that cRGD surprisingly diminished uptake in both tumor epithelial and microvascular endothelial cells by 40-50 percent in comparison to unmodified particles. Probing the mechanism of uptake revealed that the expression pattern of two isoforms of ß1 integrin impacted the uptake of cRGD-NPs differently. While the expression of high molecular weight 140 kDa form of the ß1 integrin enhanced NP uptake, the expression of low molecular 120 kDa form had an inhibitory effect. Furthermore, although, the expression of ß3 integrin was enhanced in endothelial cells and breast cancer epithelial cells, no correlation between ß3 integrin and NP uptake was observed. Additionally, in presence of clathrin and caveolae pathway inhibitors the uptake of cRGD-NPS was in general diminished with a 25-75% decrease in presence of Filipin, a caveolae inhibitor; suggesting a role for lipid rafts in the ß1 integrin-mediated uptake of cRGD-NP NPs. In sum, the polymer system described can be readily adapted to engineer other targeting peptide-based nanotherapeutics, especially for the delivery across difficult penetrate biological barriers such as the blood brain barrier. The main findings of this study have significant implication for the development of integrin targeted nanotherapeutics for anti-tumor therapy.

Renal clearance of polymeric nanoparticles by mimicry of glycan surface of viruses.

It has been shown that viral particles such as herpes simplex virus-1 and cytomegalovirus show renal clearance despite their large size (155-240 nm). Interestingly, one of the common characteristics of these viruses is their glycoprotein rich viral envelope. Since, glycosaminoglycans (GAGs) share similarities with oligosaccharide chains in the glycoproteins, we hypothesize that modification of nanoparticles (NPs) surface with naturally found GAGs could alter NP clearance characteristics by mimicking physicochemical aspects of viral glycoprotein envelope. We demonstrate that polymeric NP bearing surfaces enriched with dermatan sulfate, chondroitin sulfate, heparin sulfate, and hyaluronic acid undergo rapid renal clearance (74% of injected dose as early as 2 h) while showing reduced liver accumulation. Ultra-structural analyses suggest that the excretion of intact NPs occurs via proximal tubule secretion, but not via glomerular filtration. Finally, we demonstrate that our bioinspired NPs are able to accumulate within the epithelial tumor microenvironment despite their efficient renal clearance. Our system provides a framework to address renal toxicity associated with repeated dosing of NP and a platform to elaborate on plausible mechanism of renal clearance of virus particle.

Autophagy inhibition enhances Matrine derivative MASM induced apoptosis in cancer cells via a mechanism involving reactive oxygen species-mediated PI3K/Akt/mTOR and Erk/p38 signaling.

BACKGROUND: In the quest for new anti-cancer drugs, the drug discovery process has shifted to screening of active ingredients in traditional eastern medicine. Matrine is an active alkaloid isolated from plants of the Sophora genus used in traditional Chinese herbal medicine that exhibits a wide spectrum of biological properties and has a potential as an anti-proliferative agent. In this study, we investigated the anticancer property of MASM, ([(6aS, 10S, 11aR, 11bR, 11cS)210-Methylamino-dodecahydro-3a, 7a-diaza-benzo (de)anthracene-8-thione]), a potent derivative of matrine. METHODS: Four epithelial cancer cell lines representing the dominant cancers, namely: A549 (non-small-cell lung cancer cell line), MCF-7 and MDA-MB-231 (breast cancer cell lines), and Hela (cervical cancer cell line) were employed, and the mechanistic underpinning of MASM-induced apoptosis was investigated using flow cytometry, western blot and immunofluorescence. RESULTS: MASM, induced apoptosis via caspase 3 dependent and independent pathways, and autophagy in all the four cancer cell lines, but post-EMT (epithelial mesenchymal transition) cells showed greater sensitivity to MASM. Scavenging reactive oxygen species using N-acetylcysteine rescued all cancer cell lines from apoptosis and autophagy. Mechanistic analysis revealed that MASM induced autophagy involves inhibition of Akt signaling and the activation of Erk and p38 signaling, and inhibition of autophagy further enhanced the apoptosis induced by MASM. CONCLUSIONS: These results indicate that MASM possesses potency against cancer cells and modulating autophagy during MASM administration could be used to further enhance its therapeutic effects.

Silencing of GFP expression in human mesenchymal stem cells using quaternary polyplexes of siRNA-PEI with glycosaminoglycans and albumin.

In recent years evidence has been mounting for a role for mesenchymal stem cells (MSCs) in immunomodulation, anti-inflammatory processes, and paracrine signaling via secreted extracellular vesicles. In order to exploit these biological functions, systems to efficiently deliver genetic material into MSCs would therefore be highly desirable. In this study, efficient silencing of GFP expression by combining high N/P ratio siRNA and branched PEI (bPEI) polyplexes (siRNA-bPEI) polyplexes with glycosaminoglycans (GAGs), namely hyaluronic acid (HA), chondroitin sulfate (CS) and heparin sulfate (HS), and human serum albumin (HSA) is reported. These quaternary systems were characterized using surface charge, size and morphology and applied to MSCs, which represent a challenge due to their typically low transfection efficiency. The quaternary polyplexes promoted efficient charge shielding and release of siRNA in the cytoplasm with reduced toxicity. A high silencing efficiency of >90% (i.e., less than 10% remaining GFP expression) was achieved with noticeably reduced cellular toxicity, especially with siRNA-bPEI polyplexes modified with HA and HA + HSA. In general addition of GAGs led to more compact polyplexes. Endocytosis studies point to improved endosomal escape at high N/P ratios as a reason for high transfection efficiency and a role for hyaluronic acid in the uptake mechanism likely via CD44 interactions. Co-localization studies showed the polyplexes are stable in the cytosol over time, which correlates with a proper disassembly and subsequent silencing of GFP. Furthermore, GAG containing polyplexes were frequently co-localized with the nucleus. These findings in sum suggest that PEI/HSA/GAG based quaternary polyplexes are promising as transfection agents for MSCs. STATEMENT OF SIGNIFICANCE: Since mesenchymal stem cells (MSCs) are recruited to the site of tissue repair and play a role in immunomodulation, anti-inflammatory processes, and paracrine signaling, they present an excellent target for genetic engineering. However, delivery of genetic material into MSCs is challenging. In this study, >97% silencing of constitutive green fluorescent protein expression in human MSCs (hMSCs) using high N/P ratio polyplexes of branched-PEI-siRNA incorporating glycosaminoglycan as a charge neutralizer and human serum albumin as co-complexing agent is demonstrated. In addition to possessing good cytocompatibility and excellent cytosolic stability; polyplexes incorporating GAGs also showed altered endocytic uptake, with incorporation of hyaluronic acid promoting caveolae-mediated entry. Our system highlights the importance of physiologically derived macromolecules in delivery of genetic material into hMSCs.

Biotin-Avidin-Mediated Capture of Microspheres on Polymer Fibers.

Systems for efficient and selective capture of micro-scale objects and structures have application in many areas and are of particular relevance for selective isolation of mammalian cells. Systems for the latter should also not interfere with the biology of the cells. This study demonstrates the capture of microspheres through orthogonal coupling using biotin (ligand) and (strept)avidin (receptor). Fibrous poly(ethylene terephthalate) (PET) meshes were hydrolyzed under controlled alkaline conditions to obtain activated surfaces with COOH groups allowing for the functionalization of the PET with biotin of various spacer length. The system capture efficiency was optimized by varying the length of spacer presenting the biotin against streptavidin. In a proof of concept experiment, avidin-functionalized microspheres were used as surrogates for cells, and their capture under dynamic conditions including virous mixing and high-flow rate perfusion is demonstrated. Functionalization of PET meshes with biotin conjugated to longest spacer yielded the most efficient capture of microspheres. These preliminary results lay the foundation for the development of biosystems for capture of specific cells under physiologically relevant conditions, using biorthogonal avidin-biotin interactions.

Corrigendum to "Impact and Challenges of Mesenchymal Stem Cells in Medicine: An Overview of the Current Knowledge".

[This corrects the article DOI: 10.1155/2018/5023925.].

RGDSP functionalized carboxylated agarose as extrudable carriers for chondrocyte delivery.

The limited potential of cartilage to regenerate itself has led to development of new strategies and biomaterials for cartilage tissue engineering and regenerative medicine. Although de novo strategies for cartilage repair have been realized, extrudable hydrogels that can be administered in minimally invasive manner while simultaneously supporting chondrogenic differentiation could lead to development of new systems to deliver cells to cartilage lesions. In this work, we explored the suitability of thermo-reversible, extrudable gels derived from carboxylated agarose for maintaining human articular chondrocyte (HAC) phenotype. Towards this objective, we have investigated the impact of hydrogel stiffness and presence of integrin-binding peptide sequence GGGGRGDSP on HAC differentiation potential. We discovered that stiffer hydrogels (5.8 kPa) are more efficient than softer counterparts (0.6 kPa) in promoting chondrogenesis. Interestingly, in GGGGRGDSP modified gels, a synergy between stiffness and RGD signaling led to enhanced expression of chondrogenic related genes (aggrecan, collagen type II and sox9). These findings were also supported by quantitative analysis of sulfated glycosaminoglycans. Since carboxylated agarose are highly suitable as bioink for 3D bioprinting, we propose that extrudable GGGGRGDSP-linked stiff carboxylated agarose as a medium for direct printing of chondrocyte into cartilage lesion.

Mechanically Defined Microenvironment Promotes Stabilization of Microvasculature, Which Correlates with the Enrichment of a Novel Piezo-1+ Population of Circulating CD11b+ /CD115+ Monocytes.

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long-term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long-term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+ myeloid and CD11b+ /CD115+ monocytes that express the stretch activated cation channel Piezo-1, which is enriched prominently in the clot-like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.

Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis.

BACKGROUND: Although mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis. METHODS: Aggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-ß1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence. RESULTS: Reducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to ß-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a ß-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue. CONCLUSIONS: This study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.

Generation of 3D Soluble Signal Gradients in Cell-Laden Hydrogels Using Passive Diffusion.

Soluble signal gradients play an important role in organ patterning, cell migration, and differentiation. Currently, signal gradients in 2D cell culture are realized using microfluidics and here cells are exposed to high and nonphysiological shear stress. Tissue morphogenesis (organogenesis) however occurs in 3D and therefore there is a need for simple and practical systems to impose gradients to cells dispersed in 3D matrix. Herein, a 3D gradient generator based on passive diffusion elements that recapitulates interstitial flow and is capable of imposing predictable gradients over long length scales (6 mm) lasting up to 48 h to cells dispersed in a hydrogel environment is reported. Using recombinant human WNT3A (rhWNT3A), the spatiotemporal activation of the canonical WNT pathway in human epithelial kidney cells and human mesenchymal stems cells expressing a green fluorescence protein reporter on a transcription factor/lymphoid enhancer-binding factor (TCF/LEF) promoter is demonstrated. By refining computation models based on experimental findings, the diffusion coefficient of rhWNT3A in presence of human cells in 3D is determined. Furthermore, the formation of rhBMP4 gradients is visualized using immunohistochemistry by staining for phospho-SMAD1/5, the downstream targets of the bone morphogenetic protein (BMP) pathway. The simplicity of the gradient generator is expected to spur its adoption in studying developmental biology paradigms in vitro.

Hyperstimulation of CaSR in human MSCs by biomimetic apatite inhibits endochondral ossification via temporal down-regulation of PTH1R.

In adult bone injuries, periosteum-derived mesenchymal stem/stromal cells (MSCs) form bone via endochondral ossification (EO), whereas those from bone marrow (BM)/endosteum form bone primarily through intramembranous ossification (IMO). We hypothesized that this phenomenon is influenced by the proximity of MSCs residing in the BM to the trabecular bone microenvironment. Herein, we investigated the impact of the bone mineral phase on human BM-derived MSCs' choice of ossification pathway, using a biomimetic bone-like hydroxyapatite (BBHAp) interface. BBHAp induced hyperstimulation of extracellular calcium-sensing receptor (CaSR) and temporal down-regulation of parathyroid hormone 1 receptor (PTH1R), leading to inhibition of chondrogenic differentiation of MSCs even in the presence of chondroinductive factors, such as transforming growth factor-ß1 (TGF-ß1). Interestingly rescuing PTH1R expression using human PTH fragment (1-34) partially restored chondrogenesis in the BBHAp environment. In vivo studies in an ectopic site revealed that the BBHAp interface inhibits EO and strictly promotes IMO. Furthermore, CaSR knockdown (CaSR KD) disrupted the bone-forming potential of MSCs irrespective of the absence or presence of the BBHAp interface. Our findings confirm the expression of CaSR in human BM-derived MSCs and unravel a prominent role for the interplay between CaSR and PTH1R in regulating MSC fate and the choice of pathway for bone formation.