Yi Ting Tsai

Transplantation of mesenchymal stem cells from young donors delays aging in mice

Increasing evidence suggests that the loss of functional stem cells may be important in the aging process. Our experiments were originally aimed at testing the idea that, in the specific case of age-related osteoporosis, declining function of osteogenic precursor cells might be at least partially responsible. To test this, aging female mice were transplanted with mesenchymal stem cells from aged or young male donors. We find that transplantation of young mesenchymal stem cells significantly slows the loss of bone density and, surprisingly, prolongs the life span of old mice. These observations lend further support to the idea that age-related diminution of stem cell number or function may play a critical role in age-related loss of bone density in aging animals and may be one determinant of overall longevity.

The effect of erythropoietin on autologous stem cell-mediated bone regeneration.

Mesenchymal stem cells (MSCs) although used for bone tissue engineering are limited by the requirement of isolation and culture prior to transplantation. Our recent studies have shown that biomaterial implants can be engineered to facilitate the recruitment of MSCs. In this study, we explore the ability of these implants to direct the recruitment and the differentiation of MSCs in the setting of a bone defect. We initially determined that both stromal derived factor-1alpha (SDF-1α) and erythropoietin (Epo) prompted different degrees of MSC recruitment. Additionally, we found that Epo and bone morphogenetic protein-2 (BMP-2), but not SDF-1α, triggered the osteogenic differentiation of MSCs in vitro. We then investigated the possibility of directing autologous MSC-mediated bone regeneration using a murine calvaria model. Consistent with our in vitro observations, Epo-releasing scaffolds were found to be more potent in bridging the defect than BMP-2 loaded scaffolds, as determined by computed tomography (CT) scanning, fluorescent imaging and histological analyses. These results demonstrate the tremendous potential, directing the recruitment and differentiation of autologous MSCs has in the field of tissue regeneration.

Development of optical probes for in vivo imaging of polarized macrophages during foreign body reactions

Plasticity of macrophage (MΦ) phenotypes exist in a spectrum from classically activated (M1) cells, to alternatively activated (M2) cells, contributing to both the normal healing of tissues and the pathogenesis of implant failure. Here, folate- and mannose-based optical probes were fabricated to simultaneously determine the degree of MΦ polarization. In vitro tests show the ability of these probes to specifically target M1 and M2 cells. In an in vivo murine model, they were able to distinguish between the M1-dominated inflammatory response to infection and the M2-dominated regenerative response to particle implants. Finally, the probes were used to assess the inflammatory/regenerative properties of biomaterial implants. Our results show that these probes can be used to monitor and quantify the dynamic processes of MΦ polarization and their role in cellular responses in real time.

Real-time detection of implant-associated neutrophil responses using a formyl peptide receptor-targeting NIR nanoprobe.

Neutrophils play an important role in implant-mediated inflammation and infection. Unfortunately, current methods which monitor neutrophil activity, including enzyme measurements and histological evaluation, require many animals and cannot be used to accurately depict the dynamic cellular responses. To understand the neutrophil interactions around implant-mediated inflammation and infection it is critical to develop methods which can monitor in vivo cellular activity in real time. In this study, formyl peptide receptor (FPR)-targeting near-infrared nanoprobes were fabricated. This was accomplished by conjugating near-infrared dye with specific peptides having a high affinity to the FPRs present on activated neutrophils. The ability of FPR-targeting nanoprobes to detect and quantify activated neutrophils was assessed both in vitro and in vivo. As expected, FPR-targeting nanoprobes preferentially accumulated on activated neutrophils in vitro. Following transplantation, FPR-targeting nanoprobes preferentially accumulated at the biomaterial implantation site. Equally important, a strong relationship was observed between the extent of fluorescence intensity in vivo and the number of recruited neutrophils at the implantation site. Furthermore, FPR-targeting nanoprobes may be used to detect and quantify the number of neutrophils responding to a catheter-associated infection. The results show that FPR-targeting nanoprobes may serve as a powerful tool to monitor and measure the extent of neutrophil responses to biomaterial implants in vivo.

Real-time noninvasive monitoring of in vivo inflammatory responses using a pH ratiometric fluorescence imaging probe.

It is often difficult to continuously monitor and quantify inflammatory responses in vivo. These dynamic responses however are often accompanied by specific pH changes. A new ratiometric optical pH probe is developed by combining pH-sensitive (CypHer5E) and pH-insensitive (Oyster800) fluorescent dyes into nanoparticles for in vivo optical imaging. By taking the ratio of fluorescence intensities at different wavelengths, these nanosized sensors provide excellent measurement capabilities, and unique mapping, of the continuous in vivo pH changes for three different inflammation models. In each model a strong positive correlation is found between ratiometric pH changes and the corresponding inflammatory response measured by histological analyses. These results indicate that ratiometric imaging can provide a noninvasive, rapid, and highly sensitive optical readout for the pH-ratio changes in vivo. Furthermore this technique may be used to monitor the real-time dynamics of inflammatory processes.

In Situ Re-endothelialization via Multifunctional Nanoscaffolds

The endothelium monolayer lining in the luminal side of blood vessels provides critical antithrombotic functions. Damage to these cells will expose a highly thrombogenic subendothelium, which leads to pathological vascular changes. Using combined tissue engineering and ligand–receptor targeting strategy, we developed a biodegradable urethane-doped polyester (UPE) multifunctional targeting nanoparticle (MTN) scaffold system with dual ligands: (1) glycoprotein 1b (GP1b) to target the injured arterial endothelium and subendothelium and (2) anti-CD34 antibodies to capture endothelial progenitor cells for endothelium regeneration. The fabricated spherical MTNs of 400 nm were found to be cytocompatible and hemocompatible. Both the in vitro and ex vivo targeting of these nanoscaffolds not only showed binding specificity of MTNs onto the von Willebrand factor -coated surfaces that simulate the injured arterial walls but also competed with platelets for binding onto these injured sites. Further in vivo study has revealed that a single delivery of MTNs upon vascular injury reduced neointimal hyperplasia by 57% while increased endothelium regeneration by ∼60% in 21 days. These results support the promise of using MTN nanoscaffolds for treating vascular injury in situ.

The use of chemokine-releasing tissue engineering scaffolds in a model of inflammatory response-mediated melanoma cancer metastasis

Inflammatory responses and associated products have been implicated in cancer metastasis. However, the relationship between these two processes is uncertain due to the lack of a suitable model. Taking advantage of localized and controllable inflammatory responses induced by biomaterial implantation and the capability of tissue scaffolds to release a wide variety of chemokines, we report a novel system for studying the molecular mechanisms of inflammation-mediated cancer metastasis. The animal model is comprised of an initial subcutaneous implantation of biomaterial microspheres which prompt localized inflammatory responses, followed by the transplantation of metastatic cancer cells into the peritoneal cavity or blood circulation. Histological results demonstrated that substantial numbers of B16F10 cells were recruited to the site nearby biomaterial implants. There was a strong correlation between the degree of biomaterial-mediated inflammatory responses and number of recruited cancer cells. Inflammation-mediated cancer cell migration was inhibited by small molecule inhibitors of CXCR4 but not by neutralizing antibody against CCL21. Using chemokine-releasing scaffolds, further studies were carried out to explore the possibility of enhancing cancer cell recruitment. Interestingly, erythropoietin (EPO) releasing scaffolds, but not stromal cell-derived factor-1α-releasing scaffolds, were found to accumulate substantially more melanoma cells than controls. Rather unexpectedly, perhaps by indirectly reducing circulating cancer cells, mice implanted with EPO-releasing scaffolds had ~30% longer life span than other groups. These results suggest that chemokine-releasing scaffolds may potentially function as implantable cancer traps and serve as powerful tools for studying cancer distraction and even selective annihilation of circulating metastatic cancer cells.

Optical imaging of fibrin deposition to elucidate participation of mast cells in foreign body responses

Mast cell activation has been shown to be an initiator and a key determinant of foreign body reactions. However, there is no non-invasive method that can quantify the degree of implant-associated mast cell activation. Taking advantage of the fact that fibrin deposition is a hallmark of mast cell activation around biomaterial implants, a near infrared probe was fabricated to have high affinity to fibrin. Subsequent in vitro testing confirmed that this probe has high affinity to fibrin. Using a subcutaneous particle implantation model, we found significant accumulation of fibrin-affinity probes at the implant sites as early as 15 min following particle implantation. The accumulation of fibrin-affinity probes at the implantation sites could also be substantially reduced if anti-coagulant - heparin was administered at the implant sites. Further studies have shown that subcutaneous administration of mast cell activator - compound 48/80 - prompted the accumulation of fibrin-affinity probes. However, implant-associated fibrin-affinity probe accumulation was substantially reduced in mice with mast cell deficiency. The results show that our fibrin-affinity probes may serve as a powerful tool to monitor and measure the extent of biomaterial-mediated fibrin deposition and mast cell activation in vivo.

Multi-Ligand Poly( l -Lactic- co -Glycolic Acid) Nanoparticles Inhibit Activation of Endothelial Cells

Endothelial cell (EC) activation and inflammation is a key step in the initiation and progression of many cardiovascular diseases. Targeted delivery of therapeutic reagents to inflamed EC using nanoparticles is challenging as nanoparticles do not arrest on EC efficiently under high shear stress. In this study, we developed a novel polymeric platelet-mimicking nanoparticle for strong particle adhesion onto ECs and enhanced particle internalization by ECs. This nanoparticle was encapsulated with dexamethasone as the anti-inflammatory drug, and conjugated with polyethylene glycol, glycoprotein 1b, and trans-activating transcriptional peptide. The multi-ligand nanoparticle showed significantly greater adhesion on P-selectin, von Willebrand Factor, than the unmodified particles, and activated EC in vitro under both static and flow conditions. Treatment of injured rat carotid arteries with these multi-ligand nanoparticles suppressed neointimal stenosis more than unconjugated nanoparticles did. These results indicate that this novel multi-ligand nanoparticle is efficient to target inflamed EC and inhibit inflammation and subsequent stenosis.

Novel Thermogelling Dispersions of Polymer Nanoparticles for Controlled Protein Release

UNLABELLED A novel poly(oligo(ethylene glycol) methyl ether methacrylate-co-oligo(ethylene glycol) ethyl ether methacrylate)-poly(acrylic acid) interpenetrating network (IPN) nanoparticle was synthesized. The temperature-responsive properties of the IPN nanoparticles were investigated by a dynamic light scattering method. Atomic force microscopic images confirmed the homogenous and monodisperse morphology of the IPN nanoparticles. Both visual observation and viscosity testing demonstrated that the IPN nanoparticles exhibit thermogelling properties at body temperature, 37 °C. Subsequent studies verified that such temperature-sensitive properties of IPN nanoparticles allow their ease of injection and then slow release of model proteins, both in vitro and in vivo. Histological analysis showed that our IPN implants exerted minimal inflammation following subcutaneous implantation. Our results support the idea that, by simply mixing with proteins of interest, the novel IPN nanoparticles can be used to form in situ thermogelling devices for controlled protein release. FROM THE CLINICAL EDITOR This paper discusses a temperature responsive interpenetrating network (IPN) polymeric nanoparticle that can be used to form in situ thermogelling devices for controlled protein release by simply mixing them with proteins of interest.