Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.

Peripheral nerve regeneration remains a significant clinical challenge due to the unsatisfactory functional recovery and public health burden. Exosomes, especially those derived from mesenchymal stem cells (MSCs), are promising as potential cell-free therapeutics and gene therapy vehicles for promoting neural regeneration. In this study, we reported the differentiation of human adipose derived MSCs (hADMSCs) towards the Schwann cell (SC) phenotype (hADMSC-SCs) and then isolated exosomes from hADMSCs with and without differentiation (i.e., dExo vs uExo). We assessed and compared the effects of uExo and dExo on antioxidative, angiogenic, anti-inflammatory, and axon growth promoting properties by using various peripheral nerve-related cells. Our results demonstrated that hADMSC-SCs secreted more neurotrophic factors and other growth factors, compared to hADMSCs without differentiation. The dExo isolated from hADMSC-SCs protected rat SCs from oxidative stress and enhanced HUVEC migration and angiogenesis. Compared to uExo, dExo also had improved performances in downregulating pro-inflammatory gene expressions and cytokine secretions and promoting axonal growth of sensory neurons differentiated from human induced pluripotent stem cells. Furthermore, microRNA (miRNA) sequencing analysis revealed that exosomes and their parent cells shared some similarities in their miRNA profiles and exosomes displayed a distinct miRNA signature. Many more miRNAs were identified in dExo than in uExo. Several upregulated miRNAs, like miRNA-132-3p and miRNA-199b-5p, were highly related to neuroprotection, anti-inflammation, and angiogenesis. The dExo can effectively modulate various peripheral nerve-related cellular functions and is promising for cell-free biological therapeutics to enhance neural regeneration.

Obesity and its related health complications cause billions of dollars in healthcare costs annually in the United States, and there are yet to be safe and long-lasting anti-obesity approaches. Using brown adipose tissue (BAT) is a promising approach, as it uses fats for energy expenditure. However, the effect of the microenvironment on human thermogenic brown adipogenesis and how to generate clinically relevant sized and functioning BAT are still unknown. In our current study, we evaluated the effects of endothelial growth medium exposure on brown adipogenesis of human brown adipose progenitors (BAP). We found that pre-exposing BAP to angiogenic factors promoted brown adipogenic differentiation and metabolic activity. We further 3D bioprinted brown and white adipose progenitors within hydrogel-based bioink with controllable physicochemical properties and evaluated the cell responses in 3D bioprinted environments. We used soft, stiff, and stiff-porous constructs to encapsulate the cells. All three types had high cell viability and allowed for varying levels of function for both white and brown adipocytes. We found that the soft hydrogel constructs promoted white adipogenesis, while the stiff-porous hydrogel constructs improved both white and brown adipogenesis and were the optimal condition for promoting brown adipogenesis. Consistently, stiff-porous hydrogel constructs showed higher metabolic activities than stiff hydrogel constructs, as assessed by 2-deoxy glucose uptake (2-DOG) and oxygen consumption rate (OCR). These findings show that the physicochemical environments affect the brown adipogenesis and metabolic function, and further tuning will be able to optimize their functions. Our results also demonstrate that 3D bioprinting of brown adipose tissues with clinically relevant size and metabolic activity has the potential to be a viable option in the treatment of obesity and type 2 diabetes. One promising strategy for the treatment or prevention of obesity-mediated health complications is augmenting brown adipose tissues (BAT), which is a specialized fat that actively dissipate energy in the form of heat and maintain energy balance. In this study, we determined how pre-exposing human brown adipose progenitors (BAP) to angiogenic factors in 2D and how bioprinted microenvironments in 3D affected brown adipogenic differentiation and metabolic activity. We demonstrated that white and brown adipogenesis, and thermogenesis were regulated by tuning the bioprintable matrix stiffness and construct structure. This study not only unveils the interaction between BAP and 3D physiological microenvironments, but also presents a novel tissue engineered strategy to manage obesity and other related metabolic disorders.

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 10⁸ cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.

Plasma fibrinogen (F1) and fibronectin (pFN) polymerize to form a fibrin clot that is both a hemostatic and provisional matrix for wound healing. About 90% of plasma F1 has a homodimeric pair of γ chains (γγF1), and 10% has a heterodimeric pair of γ and more acidic γ′ chains (γγ′F1). We have synthesized a novel fibrin matrix exclusively from a 1:1 (molar ratio) complex of γγ′F1 and pFN in the presence of highly active thrombin and recombinant Factor XIII (rFXIIIa). In this matrix, the fibrin nanofibers were decorated with pFN nanoclusters (termed γγ′F1:pFN fibrin). In contrast, fibrin made from 1:1 mixture of γγF1 and pFN formed a sporadic distribution of “pFN droplets” (termed γγF1+pFN fibrin). The γγ′F1:pFN fibrin enhanced the adhesion of primary human umbilical vein endothelium cells (HUVECs) relative to the γγF1+FN fibrin. Three dimensional (3D) culturing showed that the γγ′F1:pFN complex fibrin matrix enhanced the proliferation of both HUVECs and primary human fibroblasts. HUVECs in the 3D γγ′F1:pFN fibrin exhibited a starkly enhanced vascular morphogenesis while an apoptotic growth profile was observed in the γγF1+pFN fibrin. Relative to γγF1+pFN fibrin, mouse dermal wounds that were sealed by γγ′F1:pFN fibrin exhibited accelerated and enhanced healing. This study suggests that a 3D pFN presentation on a fibrin matrix promotes wound healing.