Elastic fibers do not naturally regenerate in many proteolytic disorders, such as in abdominal aortic aneurysms, and prevent restoration of tissue homeostasis. We have shown drug-based attenuation of the stress-activated protein kinase, JNK-2 to stimulate elastic matrix neoassembly and to attenuate cellular proteolytic activity. We now investigate if JNK2 gene knockdown with small interfering RNA (siRNA) provides greater specificity of action and improved regenerative/antiproteolytic outcomes in a proteolytic injury culture model of rat aneurysmal smooth muscle cells (EaRASMCs). A siRNA dose of 12.5 nM delivered with a transfection reagent significantly enhanced downstream elastic fiber assembly and maturation versus untreated EaRASMC cultures. The optimal siRNA dose was also delivered as a complex with a polymeric transfection vector, polyethyleneimine (PEI) in preparation for future in vivo delivery. Linear 25 kDa PEI-siRNA (5:1 molar ratio of amine to phosphate) and linear 40 kDa PEI-siRNA (2.5:1 ratio) were effective in downregulating the JNK2 gene, and significantly increasing expression of elastic fiber assembly proteins, and decreases in elastolytic matrix metalloprotease-2 versus treatment controls to significantly increase mature elastic fiber assembly. The current work has identified siRNA dosing and siRNA-PEI complexing conditions that are safe and efficient in stimulating processes contributing to improved elastic matrix neoassembly via JNK2 gene knockdown. The results represent a mechanistic basis of a broader therapeutic approach to reverse elastic matrix pathophysiology in tissue disorders involving aberrations of elastic matrix homeostasis, such as in aortic aneurysms. Impact statement The elastic matrix and elastic fibers are key components of the structural extracellular matrix of elastic tissues and are essential to their stretch and recoil and to maintain healthy cell phenotype. Regeneration and repair of elastic matrix is naturally poor and impaired and is an unresolved challenge in tissue engineering. In this work, we investigate a novel gene silencing approach based on inhibiting the JNK2 gene, which provides significant downstream benefits to elastic fiber assembly and maturation. Combined with novel delivery strategies such as nanoparticles, we expect our approach to effect in situ elastic matrix repair in the future.

Tissue-engineered vascular grafts (TEVGs) require adequate extracellular matrix (ECM) to withstand arterial pressure. Tissue transglutaminase (TG2) and lysyl oxidase (LOX) are enzymes that cross-link ECM proteins and play a pivotal role in the development of vascular stiffness associated with aging. The purpose of this study is to investigate the expression of ECM cross-linking enzymes and mechanisms of scaffold degeneration leading to vascular stiffness in TEVG remodeling. Fast- and slow-degrading electrospun TEVGs were fabricated using polydioxanone (PDO) and poly(L-lactide-co-caprolactone) (PLCL) copolymer, with a PDO/PLCL ratio of 9:1 for fast-degrading and 1:1 for slow-degrading graft. These grafts were implanted in rats (n = 5/group) as abdominal aortic interposition conduits. The grafts were harvested at 1 month to evaluate patency, mechanical properties, vascular neotissue formation, and the expression of ECM cross-linking enzymes. All TEVGs were patent without any aneurysmal formation at 1 month. ECM area, TG2-positive area, and LOX-positive area were significantly greater in fast-degrading TEVGs compared to slow-degrading TEVGs, with significantly less remaining scaffold. The mechanical properties of fast-degrading TEVGs were similar to that of native aorta, as demonstrated by strain-stress curve. In conclusion, at 1 month, fast-degrading TEVGs had rapid and well-organized ECM with greater TG2 and LOX expression and native-like mechanical properties, compared to slow-degrading TEVGs. Impact statement Around 1.4 million patients in the United States require arterial prostheses each year due to cardiovascular diseases. Current synthetic vascular grafts suffer from increased risk of infection, thrombosis, a lack of endothelialization, and compliance mismatch to the native vasculature. Tissue-engineered vascular graft (TEVGs) presented in this study exhibited tunable biodegradation profiles by controlling the polymer ratio of polydioxanone/poly(L-lactide-co-caprolactone). One month after implantation, the fast-degrading TEVGs exhibited mechanical properties similar to that of native aorta, formation of endothelium, and well-organized extracellular matrix (ECM) with increased expression of tissue transglutaminase and lysyl oxidases, which are critical to the ECM remodeling process.

Research in bone tissue engineering aims to design materials that are effective at generating bone without causing significant side effects. The osteogenic potential of combining matrices and protein growth factors has been well documented, however, improvements are necessary to achieve optimal therapeutic benefits upon clinical translation. In this article, rat calvarial defects were treated with gene-activated matrices (GAMs). The GAMs used were collagen sponges mineralized with a simulated body fluid (SBF) containing a nonviral gene delivery system. Both in vitro and in vivo studies were performed to determine the optimal mode of gene delivery. After 6 weeks, the defects were extracted to assess bone formation and tissue quality through histological and microcomputed tomography analyses. The optimal GAM consisted of a collagen sponge with polyethylenimine plasmid DNA (PEI-pDNA) complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared with 19% for control samples. A follow-up in vivo study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was five parts of pBMP-2 to three parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) can optimally improve bone regeneration outcomes. Impact statement Bone tissue engineering has explored both nonviral gene delivery and the concept of biomimetic mineralization. In this study, we combined these two concepts to further enhance bone regeneration outcomes. We demonstrated that embedding polyethylenimine (PEI)-based gene delivery within a mineral layer formed from simulated body fluid (SBF) immersion can increase bone formation rates. We also demonstrated that the ratio of growth factors utilized for matrix fabrication can impact the amount of bone formed in the defect site. This research highlights a combined approach using SBF and nonviral gene delivery both in vitro and in vivo and prepares the way for future optimization of synthetic gene activated matrices.

Mast cells (MCs) are pro-inflammatory tissue-resident immune cells that play a key role in inflammation. MCs circulate in peripheral blood as progenitors and undergo terminal differentiation in the tissue microenvironment where they can remain for many years. This in situ maturation results in tissue- and species-specific MC phenotypes, culminating in significant variability in response to environmental stimuli. There are many challenges associated with studying mature tissue-derived MCs, particularly in humans. In cases where cultured MCs are able to differentiate in two-dimensional in vitro cultures, there remains an inability for full maturation. Extracellular matrix (ECM) scaffolds provide for a more physiologically relevant environment for cells in vitro and have been shown to modulate the response of other immune cells such as T cells, monocytes, and macrophages. To improve current in vitro testing platforms of MCs and to assess future use of ECM scaffolds for MC regulation, we studied the in vitro response of human MCs cultured on decellularized porcine dermis hydrogels (dermis extracellular matrix hydrogel [dECM-H]). This study investigated the effect of dECM-H on cellular metabolic activity, cell viability, and receptor expression compared to collagen type I hydrogel (Collagen-H). Human MCs showed different metabolic activity when cultured in the dECM-H and also upregulated immunoglobulin E (IgE) receptors associated with MC maturation/activation compared to collagen type I. These results suggest an overall benefit in the long-term culture of human MCs in the dECM-H compared to Collagen-H providing important steps toward a model that is more representative of in vivo conditions. Graphical abstract [Formula: see text] Impact statement Mast cells (MCs) are difficult to culture in vitro as current culture conditions and substrates fail to promote similar phenotypic features observed in vivo. Extracellular matrix (ECM)-based biomaterials offer three-dimensional, tissue-specific environments that more closely resemble in vivo conditions. Our study explores the use of dermal ECM hydrogels for MC culture and shows significant upregulation of metabolic activity, cell viability, and gene expression of markers associated with MC maturation or activation compared to collagen type I-hydrogel and tissue culture plastic controls at 7 days. These results are among the first to describe MC behavior in response to ECM hydrogels.

There is a continuous search for the ideal bioresorbable material to develop scaffolds for in situ vascular tissue engineering. As these scaffolds are exposed to the harsh hemodynamic environment during the entire transformation process from scaffold to neotissue, it is of crucial importance to maintain mechanical integrity and stability at all times. Bilayered scaffolds made of supramolecular polycarbonate-ester-bisurea were manufactured using dual electrospinning. These scaffolds contained a porous inner layer to allow for cellular infiltration and a dense outer layer to provide strength. Scaffolds (n = 21) were implanted as an interposition graft into the abdominal aorta of male Lewis rats and explanted after 1, 3, and 5 months in vivo to assess mechanical functionality and neotissue formation upon scaffold resorption. Results demonstrated conflicting graft outcomes despite homogeneity in the experimental group and scaffold production. Most grafts exhibited adverse remodeling, resulting in aneurysmal dilatation and calcification. However, a few grafts did not demonstrate such features, but instead were characterized by graft extension and smooth muscle cell proliferation in the absence of endothelium, while remaining patent throughout the study. We conclude that it remains extremely difficult to anticipate graft development and performance in vivo. Next to rational mechanical design and good performance in vitro, a thorough understanding of the mechanobiological mechanisms governing scaffold-driven arterial regeneration as well as potential influences of surgical procedures is warranted to further optimize scaffold designs. Careful analysis of the differences between preclinical successes and failures, as is done in this study, may provide initial handles for scaffold optimization and standardized surgical procedures to improve graft performance in vivo. Impact statement In situ vascular tissue engineering using cell-free bioresorbable scaffolds is investigated as an off-the-shelf option to grow small caliber arteries inside the body. In this study, we developed a bilayered electrospun supramolecular scaffold with a dense outer layer to provide mechanical integrity and a porous inner layer for cell recruitment and tissue formation. Despite homogenous scaffold properties and mechanical performance in vitro, in vivo testing as rat aorta interposition grafts revealed distinct graft outcomes, ranging from aneurysms to functional arteries. Careful analysis of this variability provided valuable insights into materials-driven in situ artery formation relevant for scaffold design and implantation procedures.

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. Impact statement Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

In the context of biointeractive dressings used for enhancing wound healing, the use of stromal vascular fraction (SVF) or adipose-derived stem cells (ASCs) hereof derived has not been fully exploited yet. Noncultured SVF, a heterogeneous mesenchymal population of cells, is attractive in the field of dermal regeneration because it can be instantaneously obtained, avoids genomic alterations, and is comparatively safer than cultured ASCs. Integra® Dermal Regeneration Template (DRT) was sprinkled with ASCs in complete medium supplemented with 10% fetal bovine serum (FBS), or SVF, obtained from emulsified or nonemulsified fat, in medium supplemented with 2% platelet-rich plasma (PRP). The presence and differentiation of cells were evaluated by standard histochemistry and immunohistochemistry, whereas conditioned media were analyzed for vascular endothelial growth factors (VEGF) by ELISA. In vitro experiments were conducted to analyze ASC proliferation in the presence of either FBS or PRP. Deposition of ASCs in medium supplemented with FBS caused their integration into Integra DRT as early as 1 h. ASCs were found as aggregates until 6-10 days without forming organized structures. When seeded onto Integra DRT, SVF cells in medium supplemented with PRP formed aggregates at early times, which at 7 and 10 days organized into vascular-like structures, lined by CD31+ and smooth muscle actin-positive cells. With nonemulsified fat, the lacunar structures did not show an organized distribution of SVF cells. PRP induced ASC proliferation although at lower level than FBS. VEGF secretion was enhanced when fat emulsification was introduced into the protocol. In conclusion, the combination of SVF cells obtained from emulsified fat, PRP, and Integra DRT exhibit synergistic effect on the formation of vessel-like structures indicating a step forward aimed at regenerative surgery for chronic wound healing.

Tissue engineering intends to create functionalized tissues/organs for regenerating the injured parts of the body using cells and scaffolds. A scaffold as a supporting substrate affects the cells' fate and behavior, including growth, proliferation, migration, and differentiation. Hydrogel as a biomimetic scaffold plays an important role in cellular behaviors and tissue repair, providing a microenvironment close to the extracellular matrix with adjustable mechanical and chemical features that can provide sufficient nutrients and oxygen. To enhance the hydrogel performance and compatibility with native niche, the cell-laden hydrogel is an attractive choice to mimic the function of the targeted tissue. Injectable hydrogels, due to the injectability, are ideal options for in vivo minimally invasive treatment. Cell-laden injectable hydrogels can be utilized for tissue regeneration in a noninvasive way. This article reviews the recent advances and future opportunities of cell-laden injectable hydrogels and their functions in tissue engineering. It is expected that this strategy allows medical scientists to develop a minimally invasive method for tissue regeneration in clinical settings. Impact statement Cell-laden hydrogels have been vastly utilized in biomedical application, especially tissue engineering. It is expected that this upcoming review article will be a motivation for the community. Although this strategy is still in its early stages, this concept is so alluring that it has attracted all scientists in the community and specialists at academic health centers. Certainly, this approach requires more development, and a bunch of crucial challenges have yet to be solved. In this review, we discuss this various aspects of this approach, the questions that must be answered, the expectations associated with it, and rational restrictions to develop injectable cell-laden hydrogels.