Tissue Engineering Part A最新文献

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. Impact Statement We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

Biological scaffold is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortages in clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix (ECM) by potentially inducing immune responses after implantation. The current study explores the fate of the decellularized (DC) scaffolds using a cocktail of chemicals following implantation without using immunosuppressants. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs. omentum) for implantation, we applied in-depth quantitative proteomics, genomics along with histology and quantitative image analysis tools to comprehensively describe and compare the proteins following DC and postimplantation. Our data helped to identify any alteration postdecullarization as well implantation. We could also monitor route-specific modulation of the ECM and regulation of the immune responses (macrophage and T cells) following implantation. The current approach opens up the possibility to monitor the fate of biological scaffolds in terms of the ECM and immune response against the implants. In addition, the identification of different routes helped us to identify differential immune responses against the implants. This study opens up the potential to identify the changes associated with chemical DC both pre- and postimplantation, which could further help to promote research in this direction. Impact Statement The development of a biological scaffold helps in the preparation of a functional organ in the clinics. In the current study, we develop a strategy for chemical decellularization and explored two different routes to understand the differential responses elicited postimplantation. The use of sensitive protein and genomic tools to study the changes creates a favorable environment for similar efforts to develop and characterize biological scaffolds before further trials in the clinics. The current study, which was carried out without any immunosuppressive agents, could help to establish (a) appropriate chemical strategies for preparing biological scaffolds as well as (b) identify putative implantable routes to circumvent any adverse immune reactions, which will ultimately decide the outcome for acceptance or rejection of the scaffold/implant.

Synthetically designed biomaterials strive to recapitulate and mimic the complex environment of natural systems. Using natural materials as a guide, the ability to create high-performance biomaterials that control cell fate, and support the next generation of cell- and tissue-based therapeutics, is starting to emerge. Supramolecular chemistry takes inspiration from the wealth of noncovalent interactions found in natural materials that are inherently complex, and using the skills of synthetic and polymer chemistry, recreates simple systems to imitate their features. Within the past decade, supramolecular biomaterials have shown utility in tissue engineering and the progress predicts a bright future. On this 30th anniversary of the Netherlands Biomaterials and Tissue Engineering society, we briefly recount the state of supramolecular biomaterials in the Dutch academic and industrial research and development context. This review provides the background, recent advances, industrial successes and challenges, as well as future directions of the field, as we see it. Throughout this work, we notice the intricate interplay between simplicity and complexity in creating more advanced solutions. We hope that the interplay and juxtaposition between these two forces can propel the field forward. Impact statement Supramolecular biomaterials based on noncovalent interactions hold the ability to rebuild some of the complexity of natural biomaterials in synthetic systems. While still in its infancy, the field is currently vigorously moving from fundamental experiments toward applications and products in the tissue engineering and regenerative medicine arena. Herein, we review the current state of the field in the Netherlands. While supramolecular biomaterials have incredible potential, systematic studies, balancing complexity and simplicity, efficient translation, and enhanced performance are all required for success of these strategies. As we move the field toward commercial solutions for clinical patients, we must also pay homage and remember the fundamental studies that allow these jumps in innovation.

Coronary artery bypass graft (CABG) surgery is an impactful treatment for coronary heart disease. Intimal hyperplasia is the central reason for the restenosis of vein grafts (VGs) after CABG. The introduction of external vascular sheaths around VGs can effectively inhibit intimal hyperplasia and ensure the patency of VGs. In this study, the well-known biodegradable copolymer poly (ɛ-caprolactone-co-l,l-lactide) (PLCL) was electrospun into high porosity external sheaths. The prednisone loaded in the PLCL sheath was slowly released during the degradation process of PLCL. Under the combined effects of sheath and prednisone, intimal hyperplasia was inhibited. For the cell experiments, all sheaths show low cytotoxicity to L929 cells at different concentrations at different time intervals. The ultrasonography and histological results showed prominent dilation and intimal hyperplasia of VG without sheath after 2 months of surgery. But there was no dilation in PLCL and PLCL_Prednisone groups. Of note, the prednisone-loaded sheath group exhibited efficacy in inhibiting intimal hyperplasia and ensured graft patency. Impact statement To inhibit intimal hyperplasia after coronary artery bypass graft, the use of external vascular sheaths can prevent vein graft (VG) dilatation, then reduce turbulent blood flow shear stress to vessel wall, and lower the stimulation of shear stress to smooth muscle cells (SMCs), so as to prevent the proliferation and migration of vascular SMC. We provide a biodegradable sheath electrospun by poly (ɛ-caprolactone-co-l,l-lactide) (PLCL) loading prednisone and utilize it around VG in animal models. Vascular ultrasound examinations show strong evidence of vascular patency. The histological alterations of VGs in PLCL_Prednisone group gave a narrower intima layer owing to the inhibition effect of prednisone.

Laminectomy can effectively decompress the spinal cord and expand the vertebral canal. However, the fibrosis that appears may cause adherence and recompression of the spinal cord or/and nerve root, which may cause failed back syndrome (FBS) and make the reexposure process more difficult. Reconstruction of the epidural fat may be an ideal method to achieve satisfactory results. Thirty-six New Zealand rabbits were randomly divided into three groups: control, extracellular matrix (ECM), and ECM+aMSCs groups. Saline, ECM gel, and ECM+aMSC complex were placed, respectively, at the fifth lumbar vertebrate of the rabbits. Epidural fat and fibrosis formation were detected by magnetic resonance imaging (MRI) and histologically at the 4th, 8th, and 12th weeks. Quantitative RT-PCR was used to detect the expression of interleukin 6 (IL-6) and transforming growth factor β (TGF-β). MRI and Oil Red O staining revealed epidural fat formation at the 12th week in the ECM+aMSCs group. Hematoxylin and eosin staining showed that the numbers of fibroblasts in the ECM gel and ECM+aMSCs groups were less than the control group at the 4th and 8th weeks (p < 0.05). Masson's trichrome staining showed that the proportion of collagen fibers in the ECM gel and ECM+aMSCs group was lower than the control group (p < 0.05). Quantitative RT-PCR showed the expressions of TGF-β and IL-6 were lower in the ECM gel and ECM+aMSCs group than those in the control group (p < 0.05) at the 4th week, but higher at the 8th week. We successfully reconstructed the epidural fat with ECM gel and aMSC complex; additionally, IL-6 and TGF-β cytokines were lower at early stage after laminectomy. Impact statement Our study revealed that epidural fat formed at the 12th week in the extracellular matrix (ECM) plus mesenchymal stem cell (MSC) group after laminectomy in rabbits; additionally, transforming growth factor β (TGF-β) (fibrosis) and interleukin 6 (IL-6) (inflammation) expression was reduced. Thus, we believe that our study makes a significant contribution to the literature because we were able to successfully reconstruct the epidural fat with an ECM gel combined with MSCs and reduce local inflammation.

Commercially available cultured epithelial keratinocyte sheets (KSs) have played an essential role in wound healing over the past four decades. Despite the initial uptake by the dermal elements, the survival rate of KS on the dermis-like tissue generated by conventional artificial dermis (AD) is low, making this method unsuitable for standard treatments. Therefore, an innovative AD such as collagen-gelatin sponge (CGS) that maintains the release of human recombinant basic fibroblast growth factor (bFGF) may promote wound healing. In this study, we examined whether combination therapy with KSs and CGS with bFGF (bFGF-CGS) could enhance KS survival by heterologous grafting by transplantation of human-derived KSs in an athymic nude rat wound model of staged skin reconstruction. The CGSs were implanted into skin defect wounds on athymic nude rats, which were then divided into two experimental groups: the bFGF group (CGSs containing bFGF, n = 8) and the control group (CGSs with saline, n = 8). Two weeks after implantation, human epithelial cell-derived KSs were grafted onto the dermis-like tissue, followed by assessment of the survival and morphology at 1 week later using digital imaging, histology (hematoxylin and eosin and Masson's trichrome staining), immunohistology (von Willebrand factor), immunohistochemistry (cytokeratin 1-5-6, Ki-67), and immunofluorescence (collagen IV, pan-cytokeratins) analyses. The bFGF group showed a significantly higher KS survival area (86 ± 58 mm² vs. 32 ± 22 mm²; p < 0.05) and increased epidermal thickness (158 ± 66 μm vs. 86 ± 40 μm; p < 0.05) compared with the control group, along with higher dermis-like tissue regeneration, neovascularization, epidermal maturation, and basement membrane development. These results indicate that the survival rate of KSs in the dermis-like tissue formed by bFGF-CGS was significantly increased. Therefore, combination treatment of bFGF-CGS and KSs shows potential for full-thickness skin defect reconstruction in clinical situations. Impact statement This study highlights how using a combination of cultures, keratinocyte sheets, and collagen-gelatin sponge containing basic fibroblast growth factors can significantly improve cell survival in athymic nude rats with staged skin reconstruction. Our study makes a significant contribution to the literature because it highlights a novel and improved strategy for treating a very common condition such as skin wounds arising from many conditions. Clinical translation of this study may be useful for treating skin wounds.

Volumetric muscle loss (VML) is the surgical or traumatic loss of skeletal muscle, which can cause loss of limb function or permanent disability. VML injuries overwhelms the endogenous regenerative capacity of skeletal muscle and results in poor functional healing outcomes. Currently, there are no approved tissue engineering treatments for VML injuries. In this study, fibrin hydrogels enriched with laminin-111 (LM-111; 50-450 μg/mL) were used for the treatment of VML of the tibialis anterior in a rat model. Treatment with fibrin hydrogel containing 450 μg/mL of LM-111 (FBN450) improved muscle regeneration following VML injury. FBN450 hydrogel treatment increased the relative proportion of contractile to fibrotic tissue as indicated by the myosin: collagen ratio on day 28 post-VML injury. FBN450 hydrogels also enhanced myogenic protein expression and increased the quantity of small to medium size myofibers (500-2000 μm²) as well as innervated myofibers. Improved contractile tissue deposition due to FBN450 hydrogel treatment resulted in a significant improvement (∼60%) in torque production at day 28 postinjury. Taken together, these results suggest that the acellular FBN450 hydrogels provide a promising therapeutic strategy for VML that is worthy of further investigation. Impact statement Muscle trauma accounts for 50-70% of total military injuries and complications involving muscle result in ∼80% of delayed amputations. The lack of a clinical standard of care for volumetric muscle loss (VML) injuries presents an opportunity to develop novel regenerative therapies and improve healing outcomes. Laminin-111-enriched fibrin hydrogel may provide a promising therapy for VML that is worthy of further investigation. The acellular nature of these hydrogels will allow for easy off the shelf access to critically injured patients and fewer regulatory hurdles during commercialization.

Once damaged, the articular cartilage has a very limited intrinsic capacity for self-renewal due to its avascular nature. If left untreated, damaged cartilage can lead to progressive degeneration of bone and eventually causes pain. Infrapatellar fat pad adipose-derived mesenchymal stromal cells (IPFP-ASCs) has a potential role for cartilage restoration. However, the therapeutic role for IPFP-ASCs remains to be evaluated in an appropriate osteochondral defect model. Thus, this study aimed to investigate the potential of using a three-dimensional (3D) cartilage construct of IPFP-ASCs as a promising source of cells to restore articular cartilage and to attenuate pain associated with the cartilage defect in an osteochondral defect model. The chondrogenic differentiation potential of the 3D cartilage construct derived from IPFP-ASCs was determined before implantation and postimplantation by gene expression and immunohistochemistry analysis. Pain-related behavior was also assessed by using a weight-bearing test. A significant pain-associated with the osteochondral defect was observed in this model in all groups postinduction; however, this pain can spontaneously resolve within 3 weeks postimplantation regardless of implantation of IPFP-ASCs constructs. The expression of SOX9 and COL2A1 genes in addition to protein expression were strongly expressed in 3D construct IPFP-ASCs. The existence of mature chondrocytes, along with significant (p < 0.05) positive immunostaining for type II collagen and aggrecan, were identified in the implanted site for up to 12 weeks compared with the untreated group, indicating hyaline cartilage regeneration. Taken together, this study demonstrated the successful outcome of osteochondral regeneration with scaffold-free IPFP-ASCs constructs in an osteochondral defect rat model. It provides novel and interesting insights into the current hypothesis that 3D construct IPFP-ASCs may offer potential benefits as an alternative approach to repair the cartilage defect. Impact statement This study provides evidence of using the human 3D scaffold-free infrapatellar fat pad adipose-derived mesenchymal stromal cells (IPFP-ASCs) construct to restore the full-thickness osteochondral defect in a rat model. This study showed that chondrogenic features of the construct could be retained for up to 12 weeks postimplantation. The results of this proof-of-concept study support that human 3D scaffold-free IPFP-ASCs construct has potential benefits in promoting the hyaline-like native cartilage restoration, which may be beneficial as a tissue-specific stem cell for cell-based cartilage therapy. There are several clinical advantages of IPFP-ASC including ease and minimal invasive harvesting, chondrogenic inducible property, and tissue-specific progenitors in the knee.

Replacement of pancreatic β-cells is one of the most promising treatment options for treatment of type 1 diabetes (T1D), even though, toxic immunosuppressive drugs are required. In this study, we aim to deliver allogeneic β-cell therapies without antirejection drugs using a bioengineered hybrid device that contains microencapsulated β-cells inside 3D polycaprolactone (PCL) scaffolds printed using melt electrospin writing (MEW). Mouse β-cell (MIN6) pseudoislets and QS mouse islets are encapsulated in alginate microcapsules, without affecting viability and insulin secretion. Microencapsulated MIN6 cells are then seeded within 3D MEW scaffolds, and these hybrid devices implanted subcutaneously in streptozotocin-treated diabetic NOD/SCID and BALB/c mice. Similar to NOD/SCID mice, blood glucose levels (BGL) are lowered from 30.1 to 4.8 mM in 25-41 days in BALB/c. In contrast, microencapsulated islets placed in prevascularized MEW scaffold 3 weeks after implantation in BALB/c mice normalize BGL (<12 mM) more rapidly, lasting for 60-105 days. The lowering of glucose levels is confirmed by an intraperitoneal glucose tolerance test. Vascularity within the implanted grafts is demonstrated and quantified by 3D-doppler ultrasound, with a linear increase over 4 weeks (r = 0.65). Examination of the device at 5 weeks shows inflammatory infiltrates of neutrophils, macrophages, and B-lymphocytes on the MEW scaffolds, but not on microcapsules, which have infrequent profibrotic walling. In conclusion, we demonstrate the fabrication of an implantable and retrievable hybrid device for vascularization and enhancing the survival of encapsulated islets implanted subcutaneously in an allotransplantation setting without immunosuppression. This study provides proof-of-concept for the application of such devices for human use, but, will require modifications to allow translation to people with T1D. Impact statement The retrievable 3D printed PCL scaffold we have produced promotes vascularization when implanted subcutaneously and allows seeded microencapsulated insulin-producing cells to normalize blood glucose of diabetic mice for at least 2 months, without the need for antirejection drugs to be administered. The scaffold is scalable for possible human use, but will require modification to ensure that normalization of blood glucose levels can be maintained long term.