Tissue Engineering Part A最新文献

The study aims to enhance the design process of tissue-engineered implants by evaluating the effects of scaffold reinforcement and cultivation conditions on extracellular matrix (ECM) development. The research investigates the hypothesis that mechanical stress drives ECM production and alignment. Furthermore, we have explored the potential of an in silico growth model to complement in vitro findings for accelerated development processes. The study employed fiber-reinforced and nonreinforced scaffolds fabricated using warp-knitted textiles and fibrin gel. Myofibroblasts embedded in the scaffolds were cultivated under static and dynamic conditions. ECM development was evaluated through mechanical testing, hydroxyproline assays, and microscopy, while an in silico growth model was used to predict ECM behavior. Static cultivation resulted in significant ECM development in both reinforced and nonreinforced samples, with nonreinforced scaffolds showing higher collagen content and alignment along the load direction. In contrast, dynamic cultivation inhibited ECM formation, potentially due to cross-contraction and washout effects. Fiber-reinforced scaffolds exhibited higher elasticity and sustained stress across cycles without structural damage. The in silico model provided valuable insights but overestimated mechanical properties due to limited validation data. Reinforced scaffolds maintained geometry and elasticity, suggesting suitability for load-bearing applications. Nonreinforced scaffolds facilitated higher ECM production but were prone to structural damage. Dynamic cultivation requires optimization, such as prestatic cultivation, to support ECM development. The combined in vitro and in silico approach offers a promising framework for scaffold design, reducing the reliance on iterative experimental processes.

Notochordal cells (NCs), abundantly found in the developing nucleus pulposus (NP), show potential for intervertebral disc regeneration because of their unique instructive and healthy matrix-producing capacity. However, NCs are lost early in life, and attempts at in vitro expansion have failed because they lose their specific phenotype. Therefore, much effort is focused on the generation of cells resembling the properties of healthy matrix-producing NP-like cells from human induced pluripotent stem cells (hiPSCs). They are considered a promising alternative for employing native NCs. Given the ongoing challenges in the field to fine-tune the differentiation protocol and obtain a high yield of mature matrix-producing cells, this study aims to build on the epigenetic memory and instructive capacity of healthy NP tissue. For this, we employed the epigenetic memory of tissue-specific hiPSCs derived from TIE2⁺ NP progenitor cells (NPPCs) and microenvironmental cues of decellularized porcine NC-derived matrix (dNCM), consisting of matrix components and bioactive factors to differentiate hiPSC into mature, healthy matrix-producing cells for NP repair. As a comparison, donor-matched minimally invasive peripheral blood mononuclear cell-derived hiPSCs were used. The results show that employing NPPC-derived hiPSCs instructed by natural cues provided by dNCM resulted in an increased expression of healthy phenotypic and matrisome-related NP markers. Furthermore, within this in vitro environment, differentiation of blood-derived hiPSC lines led to augmented differentiation into the hematopoietic and neural cell lineage. In conclusion, we demonstrate that hiPSCs derived from NPPCs achieve enhanced differentiation outcomes in the presence of dNCM, highlighting the potential impact of the epigenetic memory.

Peripheral nerve injuries (PNI) can result in significant losses of motor and sensory function. Although peripheral nerves have an innate capacity for regeneration, restoration of function after severe injury remains suboptimal. The gold standard for peripheral nerve regeneration (PNR) is autologous nerve transplantation, but this method is limited by the generation of an additional surgical site, donor-site morbidity, and neuroma formation at the site of harvest. Although targeted drug compounds have the potential to influence axonal growth, there are no drugs currently approved to treat PNI. Therefore, we propose to repurpose commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) to enhance PNR, facilitating easier clinical translation. Additionally, calcium signaling plays a crucial role in neuronal connectivity and regeneration, but how specific drugs modulate this process remains unclear. We developed an in vitro hollow channel collagen gel platform that successfully supports neuronal network formation. This study evaluated the effects of commonly used NSAIDs, namely ibuprofen and indomethacin, in our in vitro model of axonal growth, regeneration, and calcium signaling as potential treatments for PNI. Our results demonstrate enhanced axonal growth and regrowth with both ibuprofen and indomethacin, suggesting a positive influence on PNR. Further, these drugs showed enhanced calcium signaling dynamics, which we posit is a crucial aspect for nerve repair. Taken together, these findings highlight the potential of ibuprofen and indomethacin to be used as treatment options for PNI, given their dual capability to promote axonal growth and enhance calcium signaling.

Endothelial cells (ECs) play a crucial role in maintaining tissue homeostasis and functionality. Depending on their tissue of origin, ECs can be highly heterogeneous regarding their morphology, gene and protein expression, functionality, and signaling pathways. Understanding the interaction between organ-specific ECs and their surrounding tissue is therefore critical when investigating tissue homeostasis, disease development, and progression. In vitro models often lack organ-specific ECs, potentially limiting the translatability and validity of the obtained results. The goal of this study was to assess the differences between commonly used EC sources in tissue engineering applications, including human umbilical vein ECs (HUVECs), human dermal microvascular ECs (hdmvECs), and human foreskin microvascular ECs (hfmvECs), and organ-specific human pancreatic microvascular ECs (hpmvECs), and test their impact on functionality within an in vitro pancreas test system used for diabetes research. Utilizing high-resolution Raman microspectroscopy and Raman imaging in combination with established protein and gene expression analyses and exposure to defined physical signals within microfluidic cultures, we identified that ECs exhibit significant differences in their biochemical composition, relevant protein expression, angiogenic potential, and response to the application of mechanical shear stress. Proof-of-concept results showed that the coculture of isolated human islets of Langerhans with hpmvECs significantly increased the functionality when compared with control islets and islets cocultured with HUVECs. Our study demonstrates that the choice of EC type significantly impacts the experimental results, which needs to be considered when implementing ECs into in vitro models.

Autologous fat transfer is a common procedure that patients undergo to rejuvenate large soft tissue defects. However, these surgeries are complicated by limited tissue sources, donor-site morbidity, and necrosis. While the biofabrication of fat tissue can serve as a clinical option for reconstructive surgery, the influence of matrix mechanics, specifically stiffness and viscosity, on adipogenesis requires further elucidation. Additionally, the effects of these mechanical parameters on metabolic and thermogenic fat potential have yet to be investigated. In this study, gelatin methacryloyl (GelMA) polymers with varying degrees of methacrylation (DoM) were fabricated to create matrices with different stiffnesses and viscosities. Human adipose-derived mesenchymal stem cells were then encapsulated in mechanically tunable GelMA and underwent adipogenesis to investigate the effects of matrix mechanics on lipid phenotype and fat potential. Mechanical testing confirmed that GelMA stiffness was regulated by DoM and weight composition, whereas viscosity was determined by the latter. Further work revealed that while lipid phenotype became more enriched as matrix stiffness and viscosity declined, the potential toward metabolic and thermogenic fat appeared to be more viscous dependent rather than stiffness dependent. In addition, fatty acid binding protein 4 and uncoupling protein 1 gene expression exhibited viscous-dependent behavior despite comparable levels of peroxisome proliferator-activated receptor gamma. However, despite the superior role of viscosity, lipid quantity and mitochondrial abundance demonstrated stiffness-dependent behavior. Overall, this work revealed that matrix viscosity played a more superior role than stiffness in driving adipogenesis and distinguishing between metabolic and thermogenic fat potential. Ultimately, this differentiation in fat production is important for engineering ideal adipose tissue for large soft tissue defects.

Severe coronary artery disease is often treated with a coronary artery bypass graft using an autologous blood vessel. When this is not available, a commercially available synthetic graft can be used as an alternative but is associated with high failure rates and complications. Therefore, the research focus has shifted toward the development of biodegradable, regenerative vascular grafts that can convert into neoarteries. We previously developed an electrospun tropoelastin (TE)-polyglycerol sebacate (PGS) vascular graft that rapidly regenerated into a neoartery, with a cellular composition and extracellular matrix approximating the native aorta. We noted, however, that the TE-PGS graft underwent dilation until sufficient neotissue had been regenerated. This study investigated the mechanisms behind the observed dilation following TE-PGS vascular graft implantation in mice. We saw more pronounced dilation at the graft middle compared with the graft proximal and graft distal regions at 8 weeks postimplantation. Histological analysis revealed less degradation at the graft middle, although the remaining graft material appeared pitted, suggesting compromised structural and mechanical integrity. We also observed delayed cellular infiltration and extracellular matrix (ECM) deposition at the graft middle, corresponding with the area's reduced ability to resist dilation. In contrast, the graft proximal region exhibited greater degradation and significantly enhanced cellular infiltration and ECM regeneration. The nonuniform dilation was attributed to the combined effect of the regional differences in graft degradation and arterial regeneration. Consideration of these findings is crucial for graft optimization prior to its use in clinical applications.

In the present study, acellular cartilage matrix (ACM) was modified with poly-l-lysine/hyaluronic acid (PLL/HA) multilayers via detergent-enzyme chemical digestion and layer-by-layer self-assembly technology. This modified ACM was then loaded with Transforming Growth Factor Beta 3 (TGF-β3) and incorporated into a thermosensitive hydrogel (TH) to create a HA/PLL-ACM/TH composite scaffold with sustained-release function. This study aimed to evaluate the efficacy of this novel composite scaffold in promoting chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and facilitating osteochondral defect repair. In vitro, isolated, and cultured rat BMSCs were inoculated in equal amounts into TH, ACM/TH, and HA/PLL-ACM/TH groups, with or without TGF-β3 supplementation, for 21 days. Western blot (WB) analysis and immunofluorescence staining were employed to assess the expression levels of collagen II, aggrecan, and SOX-9. In vivo, osteochondral defect was created in the Sprague-Dawley rat trochlea using microdrilling. TH, ACM/TH, and HA/PLL-ACM/TH scaffolds, with or without TGF-β3, were implanted into the defect. After 6 weeks, the repairs were evaluated macroscopically, using Micro computed tomography (micro-CT), histological analysis, and immunohistochemistry. The results demonstrated that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 significantly upregulated the expression of collagen II, aggrecan, and SOX-9 compared with the control and other experimental groups. Furthermore, at 6 weeks postsurgery, the HA/PLL-ACM/TH group loaded with TGF-β3 exhibited superior tissue formation on the joint surface, as confirmed by micro-CT and histological evidence, indicating improved osteochondral repair. These findings suggest that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 holds promise as a therapeutic strategy for osteochondral defect and offers a novel approach for utilizing acellular cartilage microfilaments.

The immune system and biomaterials exhibit a well-documented synergistic interplay, essential for bone defect healing. Calcium phosphate (CaP) biomaterials, notably hydroxyapatite, β-tricalcium phosphate, and biphasic calcium phosphate, are widely employed as bone substitutes due to their inherent osteoconductivity. A key challenge for synthetic CaPs is augmenting their osteoinductive potential. Indeed, the limited translation of biomaterials into clinical practice may largely stem from insufficient immunomodulatory understanding. Current evidence reveals the complex host immune response to CaPs, which is mediated by physical and biochemical properties. Harnessing immunomodulatory strategies could bridge inflammatory modulation and osteogenesis, thereby enhancing bone regeneration. This review systematically analyzes recent advances in the molecular mechanisms of immune cell responses to CaPs during bone defect healing, deepening our understanding of immunomodulatory strategies for bone regeneration. Furthermore, key knowledge gaps are highlighted to inspire the development of spatiotemporally responsive CaPs for bone tissue engineering.

Scaffolds for bone tissue engineering have traditionally been designed to mimic the inorganic-to-organic ratio of mature bone, aiming to recapitulate its mechanical properties. However, early bone repair is not characterized by immediate mechanical strength but rather by materials that highly promote osteogenesis. In this study, we present the fabrication and evaluation of composite scaffolds composed of bioactive glass nanoparticles (BGNPs), silk fibroin, gelatin, and alginate, designed to optimize the ratio of inorganic BGNPs to biological polymers to enhance both biocompatibility and osteogenic potential. Characterization of the scaffolds revealed that the balance between BGNP and polymer content significantly influenced their structural and functional properties. Thermogravimetric analysis (TGA) showed a positive correlation between polymer content and scaffold water retention, while differential TGA(DTG) indicated that BGNPs improved the thermal stability of the polymer matrix. Swelling and biodegradation studies demonstrated that scaffolds with higher polymer content absorbed more water and degraded faster, creating a more dynamic environment conducive to cell activity. Uniaxial compression testing demonstrated that scaffolds with balanced compositions exhibited mechanical properties resembling those of the soft callus. In vitro biocompatibility tests demonstrated that scaffolds with higher polymer content were noncytotoxic, whereas those with excessive BGNPs reduced cell viability. Scaffolds with balanced compositions (Polymer blend: BGNPs = 9:1 and 7:3) showed significantly enhanced cell viability and osteogenicity, as indicated by increased alkaline phosphatase activity. Surprisingly, the optimal ratios resembled those of the soft callus, rather than mature bone. Based on these findings, we propose that scaffold designs should mimic the inorganic-to-organic composition of the soft callus, formed in the early stages of bone repair, as this composition better promotes osteogenesis. Optimizing the BGNP-to-polymer ratio is crucial for creating biomaterials that will achieve long-term clinical success.

In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0%, 10%, 20%, and 30%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, Western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprising 20% and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared with the 0% EC rings. On average, the 0%, 10%, 20%, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± standard deviation [SD]) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0%, 10%, 20%, and 30% EC rings was 88.5 ± 36. , 121 ± 59.1, 147 ± 43.1, and 206 ±  0.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue-engineered blood vessels (TEBVs) and compared with 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared with hMSCs alone and indicates a method to create a functional 3D human vascular cell coculture model. Impact Statement The goal of this work is to create an in vitro vascular model that exhibits structural and functional properties similar to those of native vascular tissue. For the first time, we demonstrated that human mesenchymal stem cells cocultured with endothelial cells as 3D cell aggregates, differentiated into smooth muscle cells, exhibited contractile protein expression, and contracted in response to endothelin-1. These tissue rings could be fused together to form cohesive tubular constructs to mimic the geometry of native vasculature. Overall, this study demonstrated a novel method to create and assess 3D human vascular tissue constructs using quantitative metrics.