The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction

To avoid complications of prosthetic vascular grafts, engineered vascular constructs have been investigated as an alternative for vascular reconstruction. The scaffolds for vascular tissue engineering remain a cornerstone of these efforts and yet many currently available options are limited by issues of inconsistency, poor adherence of vascular cells, or inadequate biomechanical properties. In this study, we investigated whether PCL/collagen scaffolds could support cell growth and withstand physiologic conditions while maintaining patency in a rabbit aortoiliac bypass model. Our results indicate that electrospun scaffolds support adherence and growth of vascular cells under physiologic conditions and that endothelialized grafts resisted adherence of platelets when exposed to blood. When implanted in vivo, these scaffolds were able to retain their structural integrity over 1 month of implantation as demonstrated by serial ultrasonography. Further, at retrieval, these scaffolds continued to maintain biomechanical strength that was comparable to native artery. This study suggests that electrospun scaffolds combined with vascular cells may become an alternative to prosthetic vascular grafts for vascular reconstruction.     

Effect of sterilization on structural and material properties of 3-D silk fibroin scaffolds

The development of porous scaffolds for tissue engineering applications requires the careful choice of properties, as these influence cell adhesion, proliferation and differentiation. Sterilization of scaffolds is a prerequisite for in vitro culture as well as for subsequent in vivo implantation. The variety of methods used to provide sterility is as diverse as the possible effects they can have on the structural and material properties of the three-dimensional (3-D) porous structure, especially in polymeric or proteinous scaffold materials. Silk fibroin (SF) has previously been demonstrated to offer exceptional benefits over conventional synthetic and natural biomaterials in generating scaffolds for tissue replacements. This study sought to determine the effect of sterilization methods, such as autoclaving, heat-, ethylene oxide-, ethanol- or antibiotic–antimycotic treatment, on porous 3-D SF scaffolds. In terms of scaffold morphology, topography, crystallinity and short-term cell viability, the different sterilization methods showed only few effects. Nevertheless, mechanical properties were significantly decreased by a factor of two by all methods except for dry autoclaving, which seemed not to affect mechanical properties compared to the native control group. These data suggest that SF scaffolds are in general highly resistant to various sterilization treatments. Nevertheless, care should be taken if initial mechanical properties are of interest.     

A porous tissue engineering scaffold selectively degraded by cell-generated reactive oxygen species

Biodegradable tissue engineering scaffolds are commonly fabricated from poly(lactide-co-glycolide) (PLGA) or similar polyesters that degrade by hydrolysis. PLGA hydrolysis generates acidic breakdown products that trigger an accelerated, autocatalytic degradation mechanism that can create mismatched rates of biomaterial breakdown and tissue formation. Reactive oxygen species (ROS) are key mediators of cell function in both health and disease, especially at sites of inflammation and tissue healing, and induction of inflammation and ROS are natural components of the in vivo response to biomaterial implantation. Thus, polymeric biomaterials that are selectively degraded by cell-generated ROS may have potential for creating tissue engineering scaffolds with better matched rates of tissue in-growth and cell-mediated scaffold biodegradation. To explore this approach, a series of poly(thioketal) (PTK) urethane (PTK-UR) biomaterial scaffolds were synthesized that degrade specifically by an ROS-dependent mechanism. PTK-UR scaffolds had significantly higher compressive moduli than analogous poly(ester urethane) (PEUR) scaffolds formed from hydrolytically-degradable ester-based diols (p < 0.05). Unlike PEUR scaffolds, the PTK-UR scaffolds were stable under aqueous conditions out to 25 weeks but were selectively degraded by ROS, indicating that their biodegradation would be exclusively cell-mediated. The in vitro oxidative degradation rates of the PTK-URs followed first-order degradation kinetics, were significantly dependent on PTK composition (p < 0.05), and correlated to ROS concentration. In subcutaneous rat wounds, PTK-UR scaffolds supported cellular infiltration and granulation tissue formation, followed first-order degradation kinetics over 7 weeks, and produced significantly greater stenting of subcutaneous wounds compared to PEUR scaffolds. These combined results indicate that ROS-degradable PTK-UR tissue engineering scaffolds have significant advantages over analogous polyester-based biomaterials and provide a robust, cell-degradable substrate for guiding new tissue formation.     

Oxygen delivery from hyperbarically loaded microtanks extends cell viability in anoxic environments

Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo.     

The development of collagen-GAG scaffold-membrane composites for tendon tissue engineering

Current tissue engineering approaches for tendon defects require improved biomaterials to balance microstructural and mechanical design criteria. Collagen-glycosaminoglycan (CG) scaffolds have shown considerable success as in vivo regenerative templates and in vitro constructs to study cell behavior. While these scaffolds possess many advantageous qualities, their mechanical properties are typically orders of magnitude lower than orthopedic tissues such as tendon. Taking inspiration from mechanically efficient core-shell composites in nature such as plant stems and porcupine quills, we have created core-shell CG composites that display high bioactivity and improved mechanical integrity. These composites feature integration of a low density, anisotropic CG scaffold core with a high density, CG membrane shell. CG membranes were fabricated via an evaporative process that allowed separate tuning of membrane thickness and elastic moduli and were found to be isotropic in-plane. The membranes were then integrated with an anisotropic CG scaffold core via freeze-drying and subsequent crosslinking. Increasing the relative thickness of the CG membrane shell was shown to increase composite tensile elastic modulus by as much as a factor of 36 in a manner consistent with predictions from layered composites theory. CG scaffold-membrane composites were found to support tendon cell viability, proliferation, and metabolic activity in vitro, suggesting they maintain sufficient permeability while demonstrating improved mechanical strength. This work suggests an effective, biomimetic approach for balancing strength and bioactivity requirements of porous scaffolds for tissue engineering.     

Tissue engineering scaffolds containing embedded fluorinated-zeolite oxygen vectors

Efficient oxygen supply is a continuing challenge for the fabrication of successful tissue engineered constructs with clinical relevance. In an effort to enhance oxygen delivery we report the feasibility of using fluorinated zeolite particles embedded in three-dimensional (3-D) polyurethane scaffolds as novel oxygen vectors. First, 1H,1H,2H,2H-perfluorodecyltriethoxysilane was successfully coupled to zeolite framework particles to examine the dose-dependent dissolved oxygen concentration. Following this, the fluorinated-zeolite (FZ) particles were embedded in 3-D tissue engineering polyurethane scaffolds. Our data demonstrates an even distribution of FZ particles in the 3-D scaffolds without affecting the scaffold porosity or pore size. Human coronary artery smooth muscle cell (HCASMC) proliferation on FZ-containing polyurethane (PCU-FZ) scaffolds was significantly greater than on control scaffolds (P = 0.05). Remarkably, cell infiltration depths on the PCU-FZ scaffolds was double that on PCU control scaffolds. Taken together, our data suggest the potential of PCU-FZ scaffolds for tissue engineering with enhanced oxygen delivery to cells.     

Effects of hydroxyapatite in 3-D chitosan-gelatin polymer network on human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have great potential in bone tissue engineering, and hydroxyapatite (HA), a natural component of human hard tissues, is believed to support hMSC growth and osteogenic differentiation. In this study, two types of biomimetic composite materials, chitosan-gelatin (CG) and hydroxyapatite/chitosan-gelatin (HCG), were fabricated and compared to examine the effects of HA on hMSC adhesion and 3-D construct development. The 2-D membranes were prepared to examine the influence of HA on adhesion efficiency of hMSCs, while 3-D porous scaffolds were produced to investigate the effects of HA on material adsorption properties and 3-D hMSC construct development. HA was found to promote protein and calcium ion adsorption of the 3-D porous scaffolds in the complete tissue culture media. HMSCs exhibited higher initial cell adhesion efficiency to 2-D HCG membranes, and maintained higher proliferation rates in the 3-D porous HCG than CG scaffolds with 3.3 times higher final DNA amount in HCG scaffolds over a 35-day period. Colony forming unit-fibroblast (CFU-F) assays showed that higher percentages of cells maintained their progenicity in the 3-D porous HCG scaffolds over the 35-day culture period. Differentiation assays indicated that the multi-lineage differentiation potential of the hMSCs was preserved in both 3-D porous scaffolds. However, higher alkaline phosphate activity was detected in the 3-D porous HCG scaffolds upon osteogenic induction indicating improved osteogenic differentiation potential. The results demonstrate that enhanced protein and calcium ion adsorption properties of HA in the CG polymer network improve initial cell adhesion and long-term growth, favor osteogenic differentiation upon induction, as well as maintain the progenicity of the 3-D hMSC constructs.     

Collagen scaffolds derived from a marine source and their biocompatibility

The primary sources of industrial collagens are calf skin and bone. However, these carry a high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy. In this study, a novel form of acid-soluble collagen was extracted from jellyfish in an effort to obtain an alternative and safer collagen. Porous scaffolds composed of jellyfish collagen were prepared by freeze-drying and cross-linking with 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide to be used in tissue engineering applications. Enzymatic degradation kinetics of jellyfish collagen scaffolds were controlled by EDC/NHS-cross-linking density. Results from an MTT assay indicated that jellyfish collagen exhibited higher cell viability than other naturally derived biomaterials, including bovine collagen, gelatin, hyaluronic acid, and glucan. Jellyfish collagen scaffolds also had a highly porous and interconnected pore structure, which is useful for an high-density cell seeding, an efficient nutrient and an oxygen supply to the cells cultured in the three-dimensional matrices. To determine whether jellyfish collagen evokes any specific inflammatory response compared to that induced by bovine collagen or gelatin, we measured the levels of pro-inflammatory cytokines and antibody secretions and monitored the population changes of immune cells after in vivo implantation. Jellyfish collagen was found to induce an immune response at least comparable to those caused by bovine collagen and gelatin.     

Nonwoven membranes for tissue engineering: an overview of cartilage,epithelium, and bone regeneration

Scaffold-type biomaterials are crucial for application in tissue engineering. Among them, the use of a nonwoven scaffold has grown in recent years and has been widely investigated for the regeneration of different types of tissues. Several polymers, whether they are synthetic, biopolymers or both, have been used to produce a scaffold that can mimic the natural tissue to which it will be applied to. The scaffolds used in tissue engineering must be biocompatible and allow cell adhesion and proliferation to be applied in tissue engineering. In addition, the scaffolds should maintain the mechanical properties and architecture of the desired tissue. Nonwoven fabrics have produced good results and are more extensively applied for the regeneration of cartilage, epithelial and bone tissues. Recent advances in tissue engineering have shown promising results, however, no ideal material or standardization parameters and characteristics of the materials were obtained. The present review provides an overview of the application of nonwoven scaffolds, including the main results obtained regarding the properties of the biomaterials and their applications in vitro and in vivo, focusing on the cartilaginous, the epithelium, and bone tissue regeneration.     

纳米材料在组织工程体外细胞培养中的应用进展

组织工程包括细胞和生物支架两部分.其中细胞的活性在很大程度上取决于支架材料的优劣.良好的生物支架应该能尽可能地模拟细胞在体内的内环境,从而提供细胞生长所需的微环境.纳米材料由于很好地模拟了细胞在体内的拓扑结构,从而在组织工程领域得到了越来越广泛的应用.综述了纳米材料在各种细胞体外培养中的作用,初步探讨了纳米材料对细胞促进作用的机制,并对纳米材料在肝脏组织工程中的应用进行了展望.     

The effect of synthetic oxygen carrier-enriched fibrin hydrogel on Schwann cells under hypoxia condition in vitro

Schwann cell (SC), which plays a key role in peripheral nerve regeneration, is one of the most classic supportive cells in neural tissue engineering. However, the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia. Thus, we aimed to investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary (48 h) oxygen deprivation. In this study, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions. The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel. SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions. The tolerance of SCs to hypoxia was examined by a cell apoptosis assay. The growth of cells was characterized using S-100 staining and a CCK-8 assay. The migration of cells was examined using a Transwell chamber. The mRNA of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), neural cell adhesion molecule (N-CAM) and vascular endothelial growth factor (VEGF) in SCs were assayed by RT-PCR. In addition, SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the mRNA levels of BDNF, NGF, GDNF, N-CAM and VEGF were assayed by RT-PCR. The results showed that the PFTBA-enriched fibrin hydrogel was able to promote cell adhesion, migration, and proliferation under hypoxic conditions. Interestingly, PFTBA applied through the fibrin hydrogel dramatically enhanced the mRNA of BDNF, NGF, GDNF, N-CAM and VEGF under hypoxic condition. These findings highlight the possibility of enhancing nerve regeneration in cellular nerve grafts through PFTBA increased neurotropic secretion in SCs.     

Biological properties of electrospun cellulose scaffolds from biomass

Abstract

Nowadays the development of sustainable technologies for the effective production of polymeric materials that can be used as biomaterials will be of importance. In this work, cellulose (CEL) was purified from potato peel waste (PPW) and used to produce electrospun nanofibers for tissue engineering applications. The purified CEL was solubilized in copper ethylenediamine (Cuen) and the electrospun nanofibers was produced through electrospinning technique in diameter range of 250–500?nm at electrical field strength of 20?kV. To confirm the applicability of the electrospun CEL scaffolds in tissue engineering, in vitro BALB-3T3 fibroblastic cell adhesion and cell proliferation tests were employed in this study. Cell viability was evaluated by staining with ethidium bromide (EtBr) and acridine orange (AO) to evaluate the possible effects of cytotoxicity of the CNF scaffolds. Fluorescence studies confirmed that BALB-3T3 viable cells attached and spread throughout the CEL scaffold. The attachment and spreading of viable cells suggests that electrospun CEL scaffolds support growth of BALB-3T3 fibroblasts cells and suggests that PPW can be a useful source of raw material for the production of scaffolds for tissue engineering.     

Biocompatibility of poly-DL-lactic acid (PDLLA) for lung tissue engineering

This study explores the possibility of growing lung cells on poly-DL-lactic acid (PDLLA) scaffolds, with a view to in future engineer pulmonary tissue for human implantation. As a first step in this process, the ability of PDLLA to maintain the growth of lung epithelium is tested using a robust cell line. Poly-DL-lactic acid has been investigated in two forms, as planar discs and as 3-D foams, and it has been demonstrated that PDLLA is not only nontoxic to pneumocytes but it also actively supports their growth. The initial findings suggest that the material is an appropriate matrix for engineering of distal lung tissue.     

In vivo bone generation via the endochondral pathway on three-dimensional electrospun fibers

A new concept of generating bone tissue via the endochondral route might be superior to the standard intramembranous ossification approach. To implement the endochondral approach, suitable scaffolds are required to provide a three-dimensional (3-D) substrate for cell population and differentiation, and eventually for the generation of osteochondral tissue. Therefore, a novel wet-electrospinning system, using ethanol as the collecting medium, was exploited in this study to fabricate a cotton-like poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffold that consisted of a very loose and uncompressed accumulation of fibers. Rat bone marrow cells were seeded on these scaffolds and chondrogenically differentiated in vitro for 4 weeks followed by subcutaneous implantation in vivo for 8 weeks. Cell pellets were used as a control. A glycosaminoglycan assay and Safranin O staining showed that the cells infiltrated throughout the scaffolds and deposited an abundant cartilage matrix after in vitro chondrogenic priming. Histological analysis of the in vivo samples revealed extensive new bone formation through the remodeling of the cartilage template. In conclusion, using the wet-electrospinning method, we are able to create a 3-D scaffold in which bone tissue can be formed via the endochondral pathway. This system can be easily processed for various assays and histological analysis. Consequently, it is more efficient than the traditional cell pellets as a tool to study endochondral bone formation for tissue engineering purposes.     

Uniformly-aligned gelatin/polycaprolactone fibers promote proliferation in adipose-derived stem cells and have distinct effects on cardiac cell differentiation

Cardiac tissue engineering is a promising technique to regenerate cardiac tissue and treat cardiovascular disease. Here we applied a modified method to generate ultrafine uniformly-aligned composite gelatin/polycaprolactone fibers that mimic functional heart tissue. We tested the physical properties of these fibers and analyzed how these composite fibrous scaffolds affected growth and cardiac lineage differentiation in rat adipose-derived stem cells (rADSCs). We found that uniformly aligned composite fiber scaffolds had an anisotropic arrangement, functional mechanical properties, and strong hydrophilicity. The anisotropic scaffolds improved cell attachment, viability, and proliferative capacity of ADSCs over randomly-aligned scaffolds. Furthermore, uniformly aligned composite fiber scaffolds increased the efficiency of cardiomyogenic differentiation, but might reduce the efficiency of cardiac conduction system cell differentiation in ADSCs compared to randomly-oriented scaffolds and tissue culture polystyrene. However, the randomly-oriented composite scaffolds showed no obviously facilitated effects over tissue culture polystyrene on the two cells’ differentiation process. The above results indicate that the scaffold fiber alignment has a greater effect on cell differentiation than the composition of the scaffold. Together, the uniformly-aligned composite fibers displayed excellent physical and biocompatible properties, promoted ADSC proliferation, and played distinct roles in the differentiation of cardiomyogenic cells and cardiac conduction system cells from ADSCs. These results provide new insight for the application of anisotropic fibrous scaffolds in cardiac tissue engineering for both in vitro and in vivo research.     

Tissue engineered bone: measurement of nutrient transport in three-dimensional matrices

The classic paradigm for in vitro tissue engineering of bone involves the isolation and culture of donor osteoblasts or osteoprogenitor cells within three-dimensional (3D) scaffold biomaterials under conditions that support tissue growth and mineralized osteoid formation. Our studies focus on the development and utilization of new dynamic culture technologies to provide adequate nutrient flux within 3D scaffolds to support ongoing tissue formation. In this study, we have developed a basic one-dimensional (1D) model to characterize the efficiency of passive nutrient diffusion and transport flux to bone cells within 3D scaffolds under static and dynamic culture conditions. Internal fluid perfusion within modeled scaffolds increased rapidly with increasing pore volume and pore diameter to a maximum of approximately 1% of external fluid flow. In contrast, internal perfusion decreased significantly with increasing pore channel tortuosity. Calculations of associated nutrient flux indicate that static 3D culture and some inappropriately designed dynamic culture environments lead to regions of insufficient nutrient concentration to maintain cell viability, and can result in steep nutrient concentration gradients within the modeled constructs. These quantitative studies provide a basis for development of new dynamic culture methodologies to overcome the limitations of passive nutrient diffusion in 3D cell-scaffold composite systems proposed for in vitro tissue engineering of bone.     

Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 μm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses.     

Development of photosynthetic biomaterials for in vitro tissue engineering

Tissue engineering has opened a new therapeutic avenue that promises a revolution in regenerative medicine. To date, however, the translation of engineered tissues into clinical settings has been highly limited and the clinical results are often disappointing. Despite decades of research, the appropriate delivery of oxygen into three-dimensional cultures still remains one of the biggest unresolved problems for in vitro tissue engineering. In this work, we propose an alternative source of oxygen delivery by introducing photosynthetic scaffolds. Here we demonstrate that the unicellular and photosynthetic microalga Chlamydomonas reinhardtii can be cultured in scaffolds for tissue repair; this microalga shows high biocompatibility and photosynthetic activity. Moreover, Chlamydomonas can be co-cultured with fibroblasts, decreasing the hypoxic response under low oxygen culture conditions. Finally, results showed that photosynthetic scaffolds are capable of producing enough oxygen to be independent of external supply in vitro. The results of this study represent the first step towards engineering photosynthetic autotrophic tissues.     

Autologous fibrin scaffolds cultured dermal fibroblasts and enriched with encapsulated bFGF for tissue engineering

Autologous fibrin scaffolds (AFSs) enriched with cells and specific growth factors represent a promising biocompatible scaffold for tissue engineering. Here, we analyzed the in vitro behavior of dermal fibroblasts (DFs) (cellular attachment, distribution, viability and proliferation, histological and immunohistochemical changes), comparing AFS with and without alginate microcapsules loaded with basic fibroblast growth factor (bFGF), to validate our scaffold in a future animal model in vivo. In all cases, DFs showed good adhesion and normal distribution, while in scaffolds with bFGF at 14 days, the cell counts detected in proliferation and viability assays were greatly improved, as was the proliferative state, and there was a decrease in muscle specific actin expression and collagen synthesis in comparison with the scaffolds without bFGF. In addition, the use of plasma without fibrinogen concentration methods, together with the maximum controlled release of bFGF at 14 days, favored cell proliferation. To conclude, we have been able to create an AFS enriched with fully functional DFs and release-controlled bFGF that could be used in multiple applications for tissue engineering.