Nano-Sized Extracellular Matrix Particles Lead to Therapeutic Improvement for Cutaneous Wound and Hindlimb Ischemia.

Cell-derived matrix (CDM) has proven its therapeutic potential and been utilized as a promising resource in tissue regeneration. In this study, we prepared a human fibroblast-derived matrix (FDM) by decellularization of in vitro cultured cells and transformed the FDM into a nano-sized suspended formulation (sFDM) using ultrasonication. The sFDM was then homogeneously mixed with Pluronic F127 and hyaluronic acid (HA), to effectively administer sFDM into target sites. Both sFDM and sFDM containing hydrogel (PH/sFDM) were characterized via immunofluorescence, sol-gel transition, rheological analysis, and biochemical factors array. We found that PH/sFDM hydrogel has biocompatible, mechanically stable, injectable properties and can be easily administered into the external and internal target regions. sFDM itself holds diverse bioactive molecules. Interestingly, sFDM-containing serum-free media helped maintain the metabolic activity of endothelial cells significantly better than those in serum-free condition. PH/sFDM also promoted vascular endothelial growth factor (VEGF) secretion from monocytes in vitro. Moreover, when we evaluated therapeutic effects of PH/sFDM via the murine full-thickness skin wound model, regenerative potential of PH/sFDM was supported by epidermal thickness, significantly more neovessel formation, and enhanced mature collagen deposition. The hindlimb ischemia model also found some therapeutic improvements, as assessed by accelerated blood reperfusion and substantially diminished necrosis and fibrosis in the gastrocnemius and tibialis muscles. Together, based on sFDM holding a strong therapeutic potential, our engineered hydrogel (PH/sFDM) should be a promising candidate in tissue engineering and regenerative medicine.

Surface functionalized magnetic nanoparticles shift cell behavior with on/off magnetic fields.

Magnetic nanoparticles (MNPs) are used as contrast agents and targeted drug delivery systems (TDDS) due to their favorable size, surface charge, and magnetic properties. Unfortunately, the toxicity associated with MNPs limits their biological applications. Surface functionalization of MNPs with selective polymers alters the surface chemistry to impart better biocompatibility. We report the preparation of surface functionalized MNPs using iron oxide NPs (MNPs), poly (lactic-co-glycolic acid) (PLGA), and sodium alginate via co-precipitation, emulsification, and electro-spraying, respectively. The NPs are in the nanosize range and negatively charged. Morphological and structural analyses affirm the surface functionalized nanostructure of the NPs. The surface functionalized MNPs are biocompatible, and demonstrate enhanced intracellular delivery under an applied magnetic field (H), which evinces the targeting ability of MNPs. After NP treatment, the physico-mechanical properties of fibroblasts are decided by the selective MNP uptake under "on" or "off" magnetic field conditions. We envision potential use of biocompatible surface functionalized MNP for intracellular-, targeted-DDS, imaging, and for investigating cellular mechanics.

Magnesium Corrosion Triggered Spontaneous Generation of H2O2 on Oxidized Titanium for Promoting Angiogenesis.

Although the use of reactive oxygen species (ROS) has been extensively studied, current systems employ external stimuli such as light or electrical energy to produce ROS, which limits their practical usage. In this report, biocompatible metals were used to construct a novel electrochemical system that can spontaneously generate H2O2 without any external light or voltage. The corrosion of Mg transfers electrons to Au-decorated oxidized Ti in an energetically favorable process, and the spontaneous generation of H2O2 in an oxygen reduction reaction was revealed to occur at titanium by combined spectroscopic and electrochemical analyses. The controlled release of H2O2 noticeably enhanced in vitro angiogenesis even in the absence of growth factors. Finally, a new titanium implant prototype was developed by Mg incorporation, and its potential for promoting angiogenesis was demonstrated.

Dual growth factor delivery using biocompatible core-shell microcapsules for angiogenesis.

An optimized electrodropping system produces homogeneous core-shell microcapsules (C-S MCs) by using poly(L-lactic-co-glycolic acid) (PLGA) and alginate. Fluorescence imaging clearly shows the C-S domain in the MC. For release control, the use of high-molecular-weight PLGA (HMW 270 000) restrains the initial burst release of protein compared to that of low-MW PLGA (LMW 40 000). Layer-by-layer (LBL) assembly of chitosan and alginate on MCs is also useful in controlling the release profile of biomolecules. LBL (7-layer) treatment is effective in suppressing the initial burst release of protein compared to no LBL (0-layer). The difference of cumulative albumin release between HMW (7-layer LBL) and LMW (0-layer LBL) PLGA is determined to be more than 40% on day 5. When dual angiogenic growth factors (GFs), such as platelet-derived GF (PDGF) and vascular endothelial GF (VEGF), are encapsulated separately in the core and shell domains, respectively, the VEGF release rate is much greater than that of PDGF, and the difference of the cumulative release percentage between the two GFs is about 30% on day 7 with LMW core PLGA and more than 45% with HMW core PLGA. As for the angiogenic potential of MC GFs with human umbilical vein endothelial cells (HUVECs), the fluorescence signal of CD31+ suggests that the angiogenic sprout of ECs is more active in MC-mediated GF delivery than conventional GF delivery, and this difference is significant, based on the number of capillary branches in the unit area. This study demonstrates that the fabrication of biocompatible C-S MCs is possible, and that the release control of biomolecules is adjustable. Furthermore, MC-mediated GFs remain in an active form and can upregulate the angiogenic activity of ECs.

Novel Corneal Endothelial Cell Carrier Couples a Biodegradable Polymer and a Mesenchymal Stem Cell-Derived Extracellular Matrix.

Here, we report a transparent, biodegradable, and cell-adhesive carrier that is securely coupled with the extracellular matrix (ECM) for corneal endothelial cell (CEC) transplantation. To fabricate a CEC carrier, poly(lactide-co-caprolactone) (PLCL) solution was poured onto the decellularized ECM (UMDM) derived from in vitro cultured umbilical cord blood-MSCs. Once completely dried, ECM-PLCL was then peeled off from the substrate. It was 20 µm thick, transparent, rich in fibronectin and collagen type IV, and easy to handle. Surface characterizations exhibited that ECM-PLCL was very rough (54.0 ± 4.50 nm) and uniformly covered in high density by ECM and retained a positive surface charge (65.2 ± 57.8 mV), as assessed via atomic force microscopy. Human CECs (B4G12) on the ECM-PLCL showed good cell attachment, with a cell density similar to the normal cornea. They could also maintain a cell phenotype, with nicely formed cell-cell junctions as assessed via ZO-1 and N-cadherin at 14 days. This was in sharp contrast to the CEC behaviors on the FNC-coated PLCL (positive control). A function-related marker, Na+/K+-ATPase, was also identified via western blot and immunofluorescence. In addition, primary rabbit CECs showed a normal shape and they could express structural and functional proteins on the ECM-PLCL. A simulation test confirmed that CECs loaded on the ECM-PLCL were successfully engrafted into the decellularized porcine corneal tissue, with a high engraftment level and cell viability. Moreover, ECM-PLCL transplantation into the anterior chamber of the rabbit eye for 8 weeks proved the maintenance of normal cornea properties. Taken together, this study demonstrates that our ECM-PLCL can be a promising cornea endothelium graft with an excellent ECM microenvironment for CECs.

Improvement of interfacial adhesion of biodegradable polymers coated on metal surface by nanocoupling.

A method of securing the adhesion of biodegradable polymer coating was investigated for drug-eluting metal stents, using surface-initiated ring-opening polymerization (SI-ROP) of L-lactide. Introduction of oligolactide on the stainless steel (SS) surface was successful and the thickness of the oligolactide grafts remained on the nanometer scale, as determined by ellipsometry. The presence of an oligolactide graft was also identified using attenuated total reflection-Fourier transform infrared (ATR-FTIR) and electron spectroscopy for chemical analysis (ESCA). On top of the grafts, poly(D,L-lactide-co-glycolide) (PLGA) coating was carried out on different substrates such as SS control, plasma-treated SS, and lactide-grafted (referred to as a nanocoupled) SS using electrospraying. When the adhesion forces were measured with a scratch tester, the nanocoupled SS showed the strongest interfacial adhesion between polymer coating layer and metal substrate. The outcome of the peel-off test was also consistent with the result of the scratch test. When degradation behavior of the polymer coating in vitro was examined for up to 4 weeks in a continuous fluid flow, the SEM images demonstrated that polymer degradation was obvious due to hydration and swelling of the polymer matrix. Although the matrix completely disappeared after 4 weeks for SS control and plasma-treated substrates, the nanocoupled SS was persistent with some polymer matrix. In addition, the release profiles of SRL-loaded PLGA coating appeared slightly different between control and nanocoupled groups. This work suggested that the concept of nanocoupling remarkably improved the interfacial adhesion stability between metal surface and polymer layer and controlled drug release, and showed the feasibility of drug-eluting stents.

Surface grafting of blood compatible zwitterionic poly(ethylene glycol) on diamond-like carbon-coated stent.

Blood compatibility is the most important aspect for blood-contacting medical devices including cardiovascular stents. In this study, the surface of nickel-titanium (TiNi) stent was coated with diamond-like carbon (DLC) and then subsequently grafted by using zwitterion (N(+) and SO(3) (-))-linked poly(ethylene glycol) (PEG). We hypothesize that this coupling of zwitterion and PEG may significantly improve blood compatibility of DLC-coated TiNi stent. The surface modified TiNi stents, including PEG-grafted stent (DLC-PEG) and zwitterionic PEG-grafted one (DLC-PEG-N-S) were the main focus on the tests of surface characteristics and blood compatibility. The zwitterionic PEG derivatives were obtained from a series of chemical reactions at room temperature. The results exhibited that as compared to the DLC-PEG, the hydrophilicity was much better with DLC-PEG-N-S and significantly increased atomic percentage of oxygen and nitrogen proved the entity of zwitterions on the surface of DLC-PEG-N-S. Meanwhile, the adsorption of blood proteins such as, human serum albumin (HSA) and fibrinogen was found considerably down-regulated in DLC-PEG-N-S, due mainly to the protein-repellent effect of PEG and zwitterion. Microscopic observation also revealed that as compared with the other substrates without zwitterion, the degree of platelet adhesion was the lowest with DLC-PEG-N-S. In addition, DLC-PEG-N-S retained an extended blood coagulation time as measured by activated partial thromboplastin time (APTT). The present results suggested that surface grafting of zwitterionic PEG derivatives could substantially enhance the blood compatibility of TiNi-DLC stent. In conclusion, anti-fouling properties of PEG and zwitterions are expected to be very useful in advancing overall stent performance.

Extracellular matrices derived from different cell sources and their effect on macrophage behavior and wound healing.

A decellularized extracellular matrix (dECM) is an excellent biomaterial in regenerative medicine, due to its biomimetic nature in targeting tissues and organs. In this study, we prepared cell-derived ECMs (CDM) derived from four different cell sources, characterized them individually, and found that intrinsic properties of each CDM were substantially different in terms of the fibrous matrix, total protein, and biochemical factors. Based on such information, we selected two ECM candidates, the human lung fibroblast derived matrix (hFDM) and the umbilical cord-blood mesenchymal stem cell derived matrix (UMDM) for the study of ECM-macrophage interactions in vitro and in vivo. In fact, UMDM was the richer in both total protein and angiogenic-related cytokines than any other CDM. When THP-1 cell-derived macrophages (M0) were seeded onto the UMDM or the hFDM, it showed a mixed cell morphology of macrophage phenotype and the macrophages (M0) preconditioned on UMDM presented more diverse cytokine release profiles. The treatment of conditioned medium obtained from CDM-seeded macrophages showed that UMDM could yield significantly advanced wound closure in 24 h via the human dermal fibroblast scratch model. To investigate the role of ECM on macrophage polarization in vivo, we prepared an ECM hydrogel, a mixture of each CDM and Pluronic F127/hyaluronan, and applied them onto a full-thickness mouse skin wound model for 2 weeks. The therapeutic efficacy as assessed via histology and immunofluorescence staining (α-SMA and CD206) revealed that the UMDM-treated group showed more effective wound healing compared to the other groups, as proven via the thinner epidermal layer, significant recovery of skin appendage, better neovascularization, and higher recruitment of myofibroblasts and larger number of macrophages (M2) at 7 days. The difference between UMDM and hFDM was marginal. Taken together, among the CDMs, UMDM and hFDM are promising resources of ECM, showing a great potential for wound healing. Although the mechanism is not fully understood, bioactive innate factors in UMDM may contribute individually and/or collectively to advance wound healing.

Novel ECM Patch Combines Poly(vinyl alcohol), Human Fibroblast-Derived Matrix, and Mesenchymal Stem Cells for Advanced Wound Healing.

Decellularized extracellular matrix (ECM)-based scaffold has been a very useful resource for effective tissue regeneration. In this study, we report a novel ECM patch that physically combines human fibroblast-derived matrix (hFDM) and poly(vinyl alcohol) (PVA) hydrogel. hFDM was obtained after decellularization of in vitro cultured human fibroblasts. We investigated the basic characteristics of hFDM alone using immunofluorescence (fibronectin, collagen type I) and angiogenesis-related factor analysis. Successful incorporation of hFDM with PVA produced an hFDM/PVA patch, which showed excellent cytocompatibility with human mesenchymal stem cells (hMSCs), as assessed via cell adhesion, viability, and proliferation. Moreover, in vitro scratch assay using human dermal fibroblasts showed a significant improvement of cell migration when treated with the paracrine factors originated from the hMSC-incorporated hFDM. To evaluate the therapeutic effect on wound healing, hMSCs were seeded on the hFDM/PVA patch and they were then transplanted into a mouse full-thickness wound model. Among four experimental groups (control, PVA, hFDM/PVA, hMSC/hFDM/PVA), we found that hMSC/hFDM/PVA patch accelerated the wound closure with time. More notably, histology and immunofluorescence demonstrated that compared to the other interventions tested, hMSC/hFDM/PVA patch could lead to significantly advanced tissue regeneration, as confirmed via nearly normal epidermis thickness, skin adnexa regeneration (hair follicle), mature collagen deposition, and neovascularization. Additionally, cell tracking of prelabeled hMSCs suggests the in vivo retention of transplanted cells in the wound region after the transplantation of hMSC/hFDM/PVA patch. Taken together, our engineered ECM patch supports a strong regenerative potential toward advanced wound healing.

Effect of chain flexibility on cell adhesion: Semi-flexible model-based analysis of cell adhesion to hydrogels.

Hydrogels have been developed and applied to various biomedical applications due to their biocompatibility. However, understanding of modulation between cells to hydrogel interface is still unclear, and parameters to explain the interaction are not sophisticated enough. In this report, we studied the effect of polymer chain flexibility on cell adhesion to various hydrogel constructs of collagen and fibrin gels. Specifically, novel method of semi-flexible model-based analysis confirmed that chain flexibility mediated microstructure of the hydrogels is a critical factor for cell adhesion on their surfaces. The proposed analysis showed possibility of more accurate prediction of biocompatibility of hydrogels, and it should be considered as one of the important criteria for polymer design and selections for enhancing both biocompatibility and biofunctionality.

Fabrication of bacterial cellulose-collagen composite scaffolds and their osteogenic effect on human mesenchymal stem cells.

Scaffold plays a critical role in stem cell differentiation and tissue regeneration. Composite scaffolds composed of bacterial cellulose (BC) and collagen (Col) in different ratios (1:1, 3:1, 5:1) were fabricated in this study. The composite scaffolds exhibit a well-organized interconnected porous structure, significantly better physical stability than Col scaffold, and more water uptake up to 400%. They were also favorable with cell attachment and growth. After osteogenic induction of umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) for 3 weeks, we found more up-regulated osteogenic markers (collagen type 1, osteocalcin, bone sialoprotein) and significantly elevated proteins and calcium deposition, particularly with BC/Col (5:1) scaffold. When PKH-26 pre-labelled MSC-loaded scaffolds were subcutaneously transplanted in a mouse model, they showed many PKH-26-labelled cells and positive signals of α-smooth muscle actin, for neovascularization in the BC/Col (5:1). The current work demonstrates that our BC/Col composites may be promising as a bone tissue-engineered scaffold.

Stretchable ECM Patch Enhances Stem Cell Delivery for Post-MI Cardiovascular Repair.

Current cell-based therapies administered after myocardial infarction (MI) show limited efficacy due to subpar cell retention in a dynamically beating heart. In particular, cardiac patches generally provide a cursory level of cell attachment due to the lack of an adequate microenvironment. From this perspective, decellularized cell-derived ECM (CDM) is attractive in its recapitulation of a natural biophysical environment for cells. Unfortunately, its weak physical property renders it difficult to retain in its original form, limiting its full potential. Here, a novel strategy to peel CDM off from its underlying substrate is proposed. By physically stamping it onto a polyvinyl alcohol hydrogel, the resulting stretchable extracellular matrix (ECM) membrane preserves the natural microenvironment of CDM, thereby conferring a biological interface to a viscoelastic membrane. Its various mechanical and biological properties are characterized and its capacity to improve cardiomyocyte functionality is demonstrated. Finally, evidence of enhanced stem cell delivery using the stretchable ECM membrane is presented, which leads to improved cardiac remodeling in a rat MI model. A new class of material based on natural CDM is envisioned for the enhanced delivery of cells and growth factors that have a known affinity with ECM.

Effect of RGD-immobilized dual-pore poly(L-lactic acid) scaffolds on chondrocyte proliferation and extracellular matrix production.

Interactions between cell and polymer surface have great implications in tissue engineering. In this study, chondrocyte proliferation and matrix production were examined using porous poly(L-lactide) (PLLA) scaffolds that have different surface characteristics. PLLA scaffolds were prepared using a gas-foaming method, and subjected to surface modifications through plasma treatment and subsequent in situ grafting of hydrophilic acrylic acid (AA). To immobilize peptide ligands, the AA-grafted PLLA scaffolds (PLLA-PAA) were further reacted with either Gly-Arg-Asp-Gly (GRDG) or Gly-Arg-Gly-Asp (GRGD) to produce PLLA-PAA-GRDG or PLLA-PAA-GRGD scaffold, respectively. The average porosities of the scaffolds were more than 90%, and their pore sizes ranged from 200 approximately 300 to 10 approximately 50 microm for large and small pores, respectively. The concentrations of each bound component were 2.14 x 10(-4) mmol/cm(2) for AA, 1.87 nmol/g for GRDG, and 1.68 nmol/g for GRGD. When chondrocytes were seeded onto the different PLLA scaffolds, cell adhesion and proliferation were highly affected as the substrate types vary. The RGD-immobilized scaffolds resulted in higher cellularity and better accumulation of total glycosaminoglycan than the others. Histological staining of Safranin O showed that the deposited extracellular matrix was more intense and widely distributed in the PLLA-PAA-GRGD scaffold. The present data suggest that immobilization of RGD peptide on the AA-grafted PLLA scaffold can be an effective tool for chondrocyte attachment and proliferation, and that it may also be helpful to facilitate cartilaginous tissue formation.

The three dimensional cues-integrated-biomaterial potentiates differentiation of human mesenchymal stem cells.

Alginate (Alg) hydrogels, the most popular natural biomaterials, mimic the extracellular matrix (ECM) microenvironment and offer potential biomedical applications. Despite their excellent properties such as biocompatibility, hydrophilicity and ionic crosslinking, the absence of an intrinsic cell adhesion domain lessens their cell-carrier applications in tissue engineering. Herein, we suggest a three-dimensional (3D) cell culture system by integrating Alg hydrogel and fibroblast-derived matrix (FDM). FDM including cell-adhesion motifs, signaling, and physico-mechanical cues is prepared by the decellularization process by avoiding unfavorable chemical reactions. This cues-integrated-biomaterials (CiB) 3D platform shows increased cell viability, proliferation, chondrogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) in situ. The results show that the Alg/FDM hydrogel (CiB) matrix provides an excellent microenvironment for cell adhesion and can control the differentiation of hMSCs into specific lineages. Thus, these results suggest the potential applications of the Alg/FDM hydrogel matrix as a viable 3D culture system for tissue regeneration.

Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing.

Skin injuries are frequently encountered in daily life, but deep wounds often poorly self-heal and do not recover completely. In this study, we propose a novel skin patch that combines antibiotic, cell-derived extracellular matrix (ECM) and biocompatible polyvinyl alcohol (PVA) hydrogel. Methods: Decellularized human lung fibroblast-derived matrix (hFDM) was prepared on tissue culture plate (TCP) and PVA solution was then poured onto it. After a freeze-thaw process, PVA was peeled off from TCP along with hFDM tightly anchored to PVA. Subsequently, ciprofloxacin (Cipro)-incorporated PVA/hFDM (PVA/Cipro/hFDM) was fabricated via diffusion-based drug loading. Results: In vitro analyses of PVA/Cipro/hFDM show little cytotoxicity of ciprofloxacin, stability of hFDM, rich fibronectin in hFDM, and good cell attachment, respectively. In addition, hFDM proved to be beneficial in promoting cell migration of dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) using transwell inserts. The antibacterial drug Cipro was very effective in suppressing colony growth of gram-negative and -positive bacteria as identified via an inhibition zone assay. For animal study, infected wound models in BALB/c mice were prepared and four test groups (control, PVA, PVA/Cipro, PVA/Cipro/hFDM) were administered separately and their effect on wound healing was examined for up to 21 days. The results support that Cipro successfully reduced bacterial infection and thus encouraged faster wound closure. Further analysis using histology and immunofluorescence revealed that the most advanced skin regeneration was achieved with PVA/Cipro/hFDM, as assessed via re-epithelialization, collagen texture and distribution in the epidermis, and skin adnexa (i.e., glands and hair follicles) regeneration in the dermis. Conclusion: This work demonstrates that our skin patch successfully consolidates the regenerative potential of ECM and the antibacterial activity of Cipro for advanced wound healing.

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber.

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly(l-lactide-co-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5-7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell-cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types.

Effects of low-intensity ultrasound on chondrogenic differentiation of mesenchymal stem cells embedded in polyglycolic acid: an in vivo study.

In this study we investigated the effects of LIUS on chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSC). Our hypothesis is that LIUS may be a noninvasively effective stimulant to a biological system in vivo by turning on differentiation of MSCs and promotion of chondrogenesis. MSCs were isolated from the bone marrow of New Zealand white rabbits and cultured in monolayer for 2 weeks. They were then harvested and seeded into polyglycolic acid (PGA) non-woven mesh at a number of 5 x 10(6) cells. Cultured with a chondrogenic-defined media for 1 week, the PGA/MSCs constructs (n = 4) were implanted subcutaneously in the back of nude mice (n = 9, each group). The ultrasound (US) group received US stimulation at a frequency of 0.8 MHz and intensity of 200 mW/cm(2) for 10 min every day up to 4 weeks, while the control group had no US stimulation. Analyses of histological, immunohistochemical, biochemical, and mechanical characteristics were made at 1, 2, and 4 weeks post-stimulation, respectively. Total DNA contents showed no significant difference between the two groups. Total collagen and glycosaminoglycan (GAG) increased more significantly in the US-stimulated group than in the control. Histology of Safranin O/Fast green confirmed more intense and spreading extracellular matrix (ECM) at 2 and 4 weeks in the US-stimulated specimens. Mechanical tests exhibited that compressive strengths were also significantly higher in the US-stimulated cells at later times. This study strongly suggests that it may be possible for ultrasound to have some stimulatory effects in vivo on the chondrogenesis of MSCs.

Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering.

Mesenchymal cells condensation is crucial in chondrogenic development. However current tissue-engineered scaffolds for chondrogenesis pay little attention to this phenomenon. In this study, we fabricate poly(l-lactide-co-glycolide) (PLGA)/poly(l-lactide) (PLLA) microfiber scaffolds and coat them with human fibroblast-derived matrix (hFDM) that is a decellularized extracellular matrix (ECM) obtained from in vitro cultured human lung fibroblasts (WI-38). Those scaffolds were then conjugated with heparin via EDC chemistry and subsequently immobilized with transforming growth factor (TGF)-ß1. The amount of TGF-ß1 was quantitatively measured and the release profile showed a continuous release of TGF-ß1 for 4 weeks. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were seeded in four different scaffolds; control, fibronectin (FN)-coated, hFDM-coated, hFDM/TGF-ß1 and subjected to chondrogenic differentiation in vitro for up to 28 days. Both hFDM and hFDM/TGF-ß1 groups exhibited significantly more synthesis of glycosaminoglycan (GAG) and much better upregulation of chondrogenic markers expression. Interestingly, MSCs condensation that led to cell aggregates was clearly observed with time in the two hFDM-coated groups and the quantitative difference was obvious compared to the control and FN group. A mechanistic study in gene and protein level indicated that focal adhesion kinase (FAK) was involved at the early stage of cell adhesion and cell-cell contact-related markers, N-cadherin and neural cell adhesion molecule (NCAM), were highly up-regulated at later time point. In addition histological analysis proved that hFDM/TGF-ß1 group was the most effective in forming neocartilage tissue in a rabbit articular cartilage defect model. Taken together, this study demonstrates not only the positive effect of hFDM on chondrogenesis of MSCs and cartilage repair but also provides an important insight toward the significance of in vitro mesenchymal condensation on chondrogenic development of MSCs.

Tunable Crosslinked Cell-Derived Extracellular Matrix Guides Cell Fate.

Extracellular matrix (ECM), comprised of multiple cues (chemical, physiomechanical), provides a niche for cell attachment, migration, and differentiation. Given that different cells give rise to distinct physiological milieus, the role of such microenvironmental cues on various cells has been well-studied. Particularly, the effect of various physiomechanical factors on stem cell lineage has been resolved into individual variables via ECM protein-coated polymeric systems. Such platforms, while providing a reductionist approach as a means to remove any confounding factors, unfortunately fall short of capturing the full biophysical scope of the natural microenvironment. Herein, the use of a cell-derived ECM platform is reported in which its crosslinking density is tunable; varying concentrations (0, 0.5, 1, 2% w/v) of genipin (GN), a naturally derived crosslinker with low toxicity, are used to form inter- and intrafibril crosslinks. ECM crosslinking produces GN concentration-dependent changes in ECM stiffness (<0.1-9.4 kPa), roughness (96-280 nm), and chemical composition (100-60% amine content). The effect of the various crosslinked ECM profiles on human mesenchymal stem cell differentiation, vascular morphogenesis, and cardiomyogenesis are then evaluated. Taken together, this study demonstrates that tunable crosslinked cell-derived ECM platform is capable of providing a comprehensive physiological platform, and envisions its use in future tissue engineering applications.

Osteogenic/angiogenic dual growth factor delivery microcapsules for regeneration of vascularized bone tissue.

Growth factors (GFs) are major biochemical cues for tissue regeneration. Herein, a novel dual GF delivery system is designed composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and alginate microcapsules (MCs) via an electrodropping method. While bone morphogenetic protein (BMP)-2 is encapsulated in the PLGA NPs, vascular endothelial growth factor (VEGF) is included in the alginate MCs, where BMP-2-loaded PLGA NPs are entrapped together in the fabrication process. The initial loading efficiencies of BMP-2 and VEGF are 78% ± 3.6% and 43% ± 1.7%, respectively. When our dual GF-loaded MCs are assessed for in vitro osteogenesis of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) on 2D and 3D environment, MCs contribute to much better UCB-MSCs osteogenesis as confirmed by von Kossa staining, immunofluorescence (osteocalcin, collagen 1), calcium content measurement, and osteogenic markers expression. In addition, when dual GF-encapsulated MCs are combined with collagen and then applied to 8 mm diameter rat calvarial defect model, the positive effects on vascularized bone regeneration are much more pronounced; micro computed tomography (CT) and histology analyses exhibit 82.3% bone healing coupled with 12.6% vessel occupied area. Put together, current study indicates a synergistic effect of BMP-2/VEGF and highlights the great potential of dual GF delivery modality (PLGA NPs-in-MC) for regeneration of vascularized bone.