Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow-/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia.

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O2) and hypoxic (5% O2) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

Compositional and structural analysis of glycosaminoglycans in cell-derived extracellular matrices.

The extracellular matrix (ECM) is a highly dynamic and complex meshwork of proteins and glycosaminoglycans (GAGs) with a crucial role in tissue homeostasis and organization not only by defining tissue architecture and mechanical properties, but also by providing chemical cues that regulate major biological processes. GAGs are associated with important physiological functions, acting as modulators of signaling pathways regulating several cellular processes such as cell growth and differentiation. Recently, in vitro fabricated cell-derived ECM have emerged as promising materials for regenerative medicine due to their ability of better recapitulate the native ECM-like composition and structure, without the limitations of availability and pathogen transfer risks of tissue-derived ECM scaffolds. However, little is known about the molecular and more specifically, GAG composition of these cell-derived ECM. In this study, three different cell-derived ECM were produced in vitro and characterized in terms of their GAG content, composition and sulfation patterns using a highly sensitive liquid chromatography-tandem mass spectrometry technique. Distinct GAG compositions and disaccharide sulfation patterns were verified for the different cell-derived ECM. Additionally, the effect of decellularization method on the GAG and disaccharide relative composition was also assessed. In summary, the method presented here offers a novel approach to determine the GAG composition of cell-derived ECM, which we believe is critical for a better understanding of ECM role in directing cellular responses and has the potential for generating important knowledge to use in the development of novel ECM-like biomaterials for tissue engineering applications.

The effect of electrospun scaffolds on the glycosaminoglycan profile of differentiating neural stem cells.

The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.

Polyaniline-polycaprolactone fibers for neural applications: Electroconductivity enhanced by pseudo-doping.

Replenishing neurons in patients with neurodegenerative diseases is one of the ultimate therapies for these progressive, debilitating and fatal diseases. Electrical stimulation can improve neuron stem cell differentiation but requires a reliable nanopatterned electroconductive substrate. Potential candidate substrates are polycaprolactone (PCL) - polyaniline:camphorsulfonic acid (PANI:CSA) nanofibers, but their nanobiophysical properties need to be finetuned. The present study investigates the use of the pseudo-doping effect on the optimization of the electroconductivity of these polyaniline-based electrospun nanofibers. This was performed by developing a new solvent system that comprises a mixture of hexafluoropropanol (HFP) and trifluoroethanol (TFE). For the first time, an electroconductivity so high as 0.2 S cm-1 was obtained for, obtained from a TFE:HFP 50/50 vol% solution, while maintaining fiber biocompatibility. The physicochemical mechanisms behind these changes were studied. The results suggest HFP promotes changes on PANI chains conformations through pseudo-doping, leading to the observed enhancement in electroconductivity. The consequences of such change in the nanofabrication of PCL-PANI fibers include an increase in fiber diameter (373 ± 172 nm), a decrease in contact angle (42 ± 3°) and a decrease in Young modulus (1.6 ± 0.5 MPa), making these fibers interesting candidates for neural tissue engineering. Electrical stimulation of differentiating neural stem cells was performed using AC electrical current. Positive effects on cell alignment and gene expression (DCX, MAP2) are observed. The novel optimized platform shows promising applications for (1) building in vitro platforms for drug screening, (2) interfaces for deep-brain electrodes; and (3) fully grown and functional neurons transplantation.

Evaluation of Autologously Derived Biomaterials and Stem Cells for Bone Tissue Engineering.

Despite progress, clinical translation of tissue engineering (TE) products/technologies is limited. A significant effort is underway to develop biomaterials and cells through a minimally modified process for clinical translation of TE products. Recently, bone marrow aspirate (BMA) was identified as an autologous source of cells for TE applications and is currently being tested in clinical therapies, but the isolation methods need improvement to avoid potential for contamination and increase progenitor cell yield. To address these issues, we reproducibly processed human peripheral blood (PB) and BMA to develop autologously derived biomaterials and cells. We demonstrated PB-derived biomaterial/gel cross-linking and fibrin gel formation with varied gelation times as well as biocompatibility through support of human bone marrow-derived stem cell survival and growth in vitro. Next, we established a plastic culture-free process that concentrates and increases the yield of CD146+/CD271+ early mesenchymal progenitor cells in BMA (concentrated BMA [cBMA]). cBMA exhibited increased colony formation and multipotency (including chondrogenic differentiation) in vitro compared with standard BMA. PB-derived gels encapsulated with cBMA also demonstrated increased cell proliferation and enhanced mineralization when assessed for bone TE in vitro. This strategy can potentially be developed for use in any tissue regeneration application; however, bone regeneration was used as a test bed for this study.

Evaluation of an Engineered Hybrid Matrix for Bone Regeneration via Endochondral Ossification.

Despite its regenerative ability, long and segmental bone defect repair remains a significant orthopedic challenge. Conventional tissue engineering efforts induce bone formation through intramembranous ossification (IO) which limits vascular formation and leads to poor bone regeneration. To overcome this challenge, a novel hybrid matrix comprised of a load-bearing polymer template and a gel phase is designed and assessed for bone regeneration. Our previous studies developed a synthetic ECM, hyaluronan (HA)-fibrin (FB), that is able to mimic cartilage-mediated bone formation in vitro. In this study, the well-characterized HA-FB hydrogel is combined with a biodegradable polymer template to form a hybrid matrix. In vitro evaluation of the matrix showed cartilage template formation, cell recruitment and recruited cell osteogenesis, essential stages in endochondral ossification. A transgenic reporter-mouse critical-defect model was used to evaluate the bone healing potential of the hybrid matrix in vivo. The results demonstrated host cell recruitment into the hybrid matrix that led to new bone formation and subsequent remodeling of the mineralization. Overall, the study developed and evaluated a novel load-bearing graft system for bone regeneration via endochondral ossification.

Kartogenin-loaded coaxial PGS/PCL aligned nanofibers for cartilage tissue engineering.

Electrospinning is a valuable technology for cartilage tissue engineering (CTE) due to its ability to produce fibrous scaffolds mimicking the nanoscale and alignment of collagen fibers present within the superficial zone of articular cartilage. Coaxial electrospinning allows the fabrication of core-shell fibers able to incorporate and release bioactive molecules (e.g., drugs or growth factors) in a controlled manner. Herein, we used coaxial electrospinning to produce coaxial poly(glycerol sebacate) (PGS)/poly(caprolactone) (PCL) aligned nanofibers (core:PGS/shell:PCL). The obtained scaffolds were characterized in terms of their structure, chemical composition, thermal properties, mechanical performance and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. All the electrospun scaffolds produced presented average fiber diameters within the nanometer-scale and the core-shell structure of the composite fibers was clearly confirmed by TEM. Additionally, fiber alignment significantly increased (>2-fold) the elastic modulus of both coaxial and monoxial scaffolds. Kartogenin (KGN), a small molecule known to promote mesenchymal stem/stromal cells (MSC) chondrogenesis, was loaded into the core PGS solution to generate coaxial PGS-KGN/PCL nanofibers. The KGN release kinetics and scaffold biological performance were evaluated in comparison to KGN-loaded monoaxial fibers and respective non-loaded controls. Coaxial PGS-KGN/PCL nanofibers showed a more controlled and sustained KGN release over 21 days than monoaxial PCL-KGN nanofibers. When cultured with human bone marrow MSC in incomplete chondrogenic medium (without TGF-ß3), KGN-loaded scaffolds enhanced significantly cell proliferation and chondrogenic differentiation, as suggested by the increased sGAG amounts and chondrogenic markers gene expression levels. Overall, these findings highlight the potential of using coaxial PGS-KGN/PCL aligned nanofibers as a bioactive scaffold for CTE applications.

Hydroporator: a hydrodynamic cell membrane perforator for high-throughput vector-free nanomaterial intracellular delivery and DNA origami biostability evaluation.

The successful intracellular delivery of exogenous macromolecules is crucial for a variety of applications ranging from basic biology to the clinic. However, traditional intracellular delivery methods such as those relying on viral/non-viral nanocarriers or physical membrane disruptions suffer from low throughput, toxicity, and inconsistent delivery performance and are time-consuming and/or labor-intensive. In this study, we developed a single-step hydrodynamic cell deformation-induced intracellular delivery platform named "hydroporator" without the aid of vectors or a complicated/costly external apparatus. By utilizing only fluid inertia, the platform focuses, guides, and stretches cells robustly without clogging. This rapid hydrodynamic cell deformation leads to both convective and diffusive delivery of external (macro)molecules into the cell through transient plasma membrane discontinuities. Using this hydroporation approach, highly efficient (∼90%), high-throughput (>1 600 000 cells per min), and rapid delivery (∼1 min) of different (macro)molecules into a wide range of cell types was achieved while maintaining high cell viability. Taking advantage of the ability of this platform to rapidly deliver large molecules, we also systematically investigated the temporal biostability of vanilla DNA origami nanostructures in living cells for the first time. Experiments using two DNA origami (tube- and donut-shaped) nanostructures revealed that these nanostructures can maintain their structural integrity in living cells for approximately 1 h after delivery, providing new opportunities for the rapid characterization of intracellular DNA biostability.

Remodeling of Glycosaminoglycans During Differentiation of Adult Human Bone Mesenchymal Stromal Cells Toward Hepatocytes.

There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.

POLYBENZIMIDAZOLE NANOFIBERS FOR NEURAL STEM CELL CULTURE.

Neurodegenerative diseases compromise the quality of life of increasing numbers of the world's aging population. While diagnosis is possible no effective treatments are available. Strong efforts are needed to develop new therapeutic approaches, namely in the areas of tissue engineering and deep brain stimulation (DBS). Conductive polymers are the ideal material for these applications due to the positive effect of conducting electricity on neural cell's differentiation profile. This novel study assessed the biocompatibility of polybenzimidazole (PBI), as electrospun fibers and after being doped with different acids. Firstly, doped films of PBI were used to characterize the materials' contact angle and electroconductivity. After this, fibers were electrospun and characterized by SEM, FTIR and TGA. Neural Stem Cell's (NSC) proliferation was assessed and their growth rate and morphology on different samples was determined. Differentiation of NSCs on PBI - CSA fibers was also investigated and gene expression (SOX2, NES, GFAP, Tuj1) was assessed through Immunochemistry and qPCR. All the samples tested were able to support neural stem cell (NSC) proliferation without significant changes on the cell's typical morphology. Successfully differentiation of NSCs towards neural cells on PBI - CSA fibers was also achieved. This promising PBI fibrous scaffold material is envisioned to be used in neural cell engineering applications, including scaffolds, in vitro models for drug screening and electrodes.

Hybrid extracellular matrix design for cartilage-mediated bone regeneration.

Recapitulating long bone repair through endochondral ossification (EO) is increasingly becoming a more popular approach. A successful EO Process depends greatly on the establishment of a healthy hypertrophic-cartilage template (HCT). The aim of this work is to design a hydrogel system, which closely mimics the extracellular matrix of HCT. We examined the combinatorial effect of two commonly used hydrogels for bone and cartilage regeneration strategies, hyaluronan (HA) and fibrin (FB), to induce HCT formation. Hydrogel combinations were evaluated using a clinically relevant cell source, human bone marrow mesenchymal stem cells (hBMSCs). The results establish that with increasing HA (50-90%) the chondrogenic and its subsequent hypertrophy trend improved, with 70:30 HA:FB combination showing the highest and most uniform expression of chondrogenic and hypertrophic stage specific markers. This combination also showed superior support for cell micro-aggregation and differentiation. Thus, 70:30 HA-FB matrix demonstrated a healthy formation of chondrogenic and hypertrophic stages with rich stage-specific ECM components. This study demonstrates that with the appropriate hydrogel design it is possible to develop effective tissue engineering therapies for bone defect repair and regeneration through endochondral ossification by establishing a healthy HCT. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 300-309, 2018.

Biodegradable and Bioactive PCL-PGS Core-Shell Fibers for Tissue Engineering.

Poly(glycerol sebacate) (PGS) has increasingly become a desirable biomaterial due to its elastic mechanical properties, biodegradability, and biocompatibility. Here, we report microfibrous core-shell mats of polycaprolactone (PCL)-PGS prepared using wet-wet coaxial electrospinning. The anticoagulant heparin was immobilized onto the surface of these electrospun fiber mats, and they were evaluated for their chemical, mechanical, and biological properties. The core-shell structure of PCL-PGS provided tunable degradation and mechanical properties. The slowly degrading PCL provided structural integrity, and the fast degrading PGS component increased fiber elasticity. Young's modulus of PCL-PGS ranged from 5.6 to 15.7 MPa. The ultimate tensile stress ranged from 2.0 to 2.9 MPa, and these fibers showed elongation from 290 to 900%. The addition of PGS and grafting of heparin improved the attachment and proliferation of human umbilical vein endothelial cells. Core-shell PCL-PGS fibers demonstrate improved performance as three-dimensional fibrous mats for potential tissue-engineering applications.

Design, fabrication and in vitro evaluation of a novel polymer-hydrogel hybrid scaffold for bone tissue engineering.

The development of a bone mechanically-compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically-sized bone defects. Although previous studies with weight-bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold-based bone regeneration. In this study, we designed and fabricated a novel polymer-hydrogel hybrid scaffold system in which a load-bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre-osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin-streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre-osteoblast cell line MC3T3-E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx-2 and osteocalcin (OC) increased in MC3T3-E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer-hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering.

A potential translational approach for bone tissue engineering through endochondral ossification.

Bone defect repair is a significant clinical challenge in orthopedic surgery. Despite tremendous efforts, the majority of the current bone tissue engineering strategies depend on bone formation via intramembranous ossification (IO), which often results in poor vascularization and limited-area bone regeneration. Recently, there has been increasing interest in exploring bone regeneration through a cartilage-mediated process similar to endochondral ossification (EO). This method is advantageous because long bones are originally developed through EO and moreover, vascularization is an inherent step of this process. Therefore, it may be possible to effectively employ the EO method for the repair and regeneration of large and segmental bone defects. Although a number of studies have demonstrated engineered bone formation through EO, there are no approaches aiming for their clinical translation. In this study, we propose a strategy modeled after the U.S. Food and Drug Administration (FDA) approved autologus chondrocyte implantation (ACI) procedure. In its implementation, we concentrated human bone marrow aspirate via a minimally manipulated process and demonstrated the potential of human bone marrow derived cells for in vitro pre-cartilage template formation and bone regeneration in vivo.

Functionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: fabrication, in vitro and in vivo evaluation.

Designing biodegradable scaffolds with bone-compatible mechanical properties has been a significant challenge in the field of bone tissue engineering and regenerative engineering. The objective of this work is to improve the polymeric scaffold's mechanical strength by compositing it with mechanically superior carbon nanotubes. Poly(lactide-co-glycolide) (PLGA) microsphere scaffolds exhibit mechanical properties in the range of human cancellous bone. On the other hand, carbon nanotubes have outstanding mechanical properties. The aim of this study is to improve further the mechanical strength of PLGA scaffolds such that they may be applicable for a wide range of load-bearing repair and regeneration applications. We have formed composite microspheres of PLGA containing pristine and modified (with hydroxyl (OH), carboxylic acid (COOH)) multi-walled carbon nanotubes (MWCNTs), and fabricated them into three-dimensional porous scaffolds. Results show that by adding only 3% MWCNTs, the compressive strength and modulus was significantly increased (35 MPa, 510.99 MPa) compared to pure PLGA scaffolds (19 MPa and 166.38 MPa). Scanning electron microscopy images showed excellent cell adhesion and proliferation. In vitro studies exhibited good cell viability, proliferation and mineralization. The in vivo study, however, indicated differences in inflammatory response throughout the 12 weeks of implantation, with OH-modified MWCNTs having the least response, followed by unmodified and COOH-modified exhibiting a more pronounced response. Overall, our results show that PLGA scaffolds containing water-dispersible MWCNTs are mechanically stronger and display good cellular and tissue compatibility, and hence are potential candidates for load-bearing bone tissue engineering.