Fabrication of gelatin-hyaluronic acid hybrid scaffolds with tunable porous structures for soft tissue engineering

The development of three-dimensional (3-D) scaffolds with highly open porous structure is one of the most important issues in tissue engineering. In this study, 3-D macroporous gelatin/hyaluronic acid (GE/HA) hybrid scaffolds with varying porous morphology were prepared by freeze-drying their blending solutions and subsequent chemical crosslinking by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The resulting scaffolds were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Their swelling, in vitro degradation properties and compressive strength were also investigated. To evaluate in vitro cytocompatibility of scaffolds, mouse L929 fibroblasts were seeded onto the scaffolds for cell morphology and cell viability studies. It was found that the porous structure of scaffolds can be tailored by varying the ratios of gelatin to HA, both the swelling ratios and degradation rate increased with the increase of HA content in hybrid scaffolds, and crosslinking the scaffolds with EDC improved the degradation resistance of the scaffold in culture media and increased the mechanical strength of scaffolds. The in vitro results revealed that the prepared scaffolds do not induce cytotoxic effects and suitable for cell growth, especially in the case of scaffolds with higher gelatin content. The combined results of the physicochemical and biological studies suggested that the developed GE/HA hybrid scaffolds exhibit good potential and biocompatibility for soft tissue engineering applications.     

Supercritical fluid-assisted controllable fabrication of open and highly interconnected porous scaffolds for bone tissue engineering

Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone(PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 2~3 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2(BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells(BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase(ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.     

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.

We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.     

Electrospun synthetic and natural nanofibers for regenerative medicine and stem cells

Nanofibers are attractive substrates for tissue regeneration applications because they structurally mimic the native extracellular matrix. Electrospinning has been recognized as one of the most efficient techniques to fabricate polymer nanofibers. Recent research has demonstrated that cellular responses, for example attachment, proliferation and differentiation, can be modulated by tuning nanofiber properties. In combination with other processing techniques, such as particulate leaching or three-dimensional printing, nanofibrous scaffolds incorporating macroporous networks could be developed to enhance infiltration of cells. Three dimensional nanofiber-based constructs offer an opportunity to achieve advanced functional tissue regeneration. This review explores the advantageous effects of nanofibers on cell behaviors compared to traditional scaffolds.     

Fabrication of individual scaffolds based on a patient-specific alveolar bone defect model

Fabricating individualized tissue engineering scaffolds based on the three-dimensional shape of patient bone defects is required for the successful clinical application of bone tissue engineering. However, there are currently no reported studies of individualized bone tissue engineering scaffolds that truly reproduce a patient-specific bone defect. We fabricated individualized tissue engineering scaffolds based on alveolar bone defects. The individualized poly(lactide-co-glycolide) and tricalcium phosphate composite scaffolds were custom-made by acquiring the three-dimensional model through computed tomography, which was input into the computer-aided low-temperature deposition manufacturing system. The three-dimensional shape of the fabricated scaffold was identical to the patient-specific alveolar bone defects, with an average macropore diameter of 380 μm, micropore diameters ranging from 3 to 5 μm, and an average porosity of 87.4%. The mechanical properties of the scaffold were similar to adult cancellous bone. Scaffold biocompatibility was confirmed by attachment and proliferation of human bone marrow mesenchymal stem cells. Successful realization of individualized scaffold fabrication will enable clinical application of tissue-engineered bone at an early date.     

Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation

One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three-dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long-term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non-woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30-day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: (1) free-swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage.     

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.     

The development and identification of constructing tissue engineered bone by seeding osteoblasts from differentiated rat marrow stromal stem cells onto three-dimensional porous nano-hydroxylapatite bone matrix in vitro

The purposes of this study were to develop a new cultural method for the rat bone marrow stromal cells (MSCs) to differentiate into osteoblasts well in vitro, and to investigate the feasibility of using MSCs as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds for constructing tissue-engineered bone. MSCs of rats were isolated, cultured, induced to differentiate into osteoblasts, and then observed with inverted microscopy. Histochemical staining and radio-immunological analysis were applied for identifying MSCs. Whereafter MSCs were seeded onto three-dimensional porous nano-hydroxylapatite scaffolds, and scanning electron microscopy was applied to evaluate their growth on scaffolds. Results showed that MSCs were typical fibroblast-like and possessed a better proliferating capability; the activity of alkaline phosphatase (ALP) and the secretion of osteocalcin of MSCs were produced gradually and increased continuously; the cells seeded on three-dimensional porous nano-hydroxylapatite scaffolds adhered, proliferated and differentiated well. These results demonstrated that the new improved culture method had the advantages of short isolating time, less risk of contamination and higher efficiency and accordingly was conducive to MSCs proliferating and differentiating into osteoblasts, and that it was advantageous to constructing tissue-engineered bone using MSCs as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds.     

Engineered Smooth Muscle Tissues: Regulating Cell Phenotype with the Scaffold

Culturing cells on three-dimensional, biodegradable scaffolds may create tissues suitable either for reconstructive surgery applications or as novel in vitro model systems. In this study, we have tested the hypothesis that the phenotype of smooth muscle cells (SMCs) in three-dimensional, engineered tissues is regulated by the chemistry of the scaffold material. Specifically, we have directly compared cell growth and patterns of extracellular matrix (ECM) (e.g. , elastin and collagen) gene expression on two types of synthetic polymer scaffolds and type I collagen scaffolds. The growth rates of SMCs on the synthetic polymer scaffolds were significantly higher than on type I collagen sponges. The rate of elastin production by SMCs on polyglycolic acid (PGA) scaffolds was 3.5 +/- 1.1-fold higher than that on type I collagen sponges on Day 11 of culture. In contrast, the collagen production rate on type I collagen sponges was 3.3 +/- 1.1-fold higher than that on PGA scaffolds. This scaffold-dependent switching between elastin and collagen gene expression was confirmed by Northern blot analysis. The finding that the scaffold chemistry regulates the phenotype of SMCs independent of the scaffold physical form was confirmed by culturing SMCs on two-dimensional films of the scaffold materials. It is likely that cells adhere to these scaffolds via different ligands, as the major protein adsorbed from the serum onto synthetic polymers was vitronectin, whereas fibronectin and vitronectin were present at high density on type I collagen sponges. In summary, this study demonstrates that three-dimensional smooth muscle-like tissues can be created by culturing SMCs on three-dimensional scaffolds, and that the phenotype of the SMCs is strongly regulated by the scaffold chemistry. These engineered tissues provide novel, three-dimensional models to study cellular interaction with ECM in vitro.     

Monosaccharide-Responsive Phenylboronate-Polyol Cell Scaffolds for Cell Sheet and Tissue Engineering Applications

Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.     

Biodegradable fumarate-based polyHIPEs as tissue engineering scaffolds

PolyHIPEs show great promise as tissue engineering scaffolds due to the tremendous control of pore size and interconnectivity afforded by this technique. Highly porous, fully biodegradable scaffolds were prepared by polymerization of the continuous phase of high internal phase emulsions (HIPEs) containing the macromer poly(propylene fumarate) (PPF) and the cross-linker propylene fumarate diacrylate (PFDA). Toluene was used as a diluent to reduce the viscosity of the organic phase to enable HIPE formation. A range of polyHIPE scaffolds of different pore sizes and morphologies were generated by varying the diluent concentration (40-60 wt %), cross-linker concentration (25-75 wt %), and macromer molecular weight ( M n = 800-1000 g/mol). Although some formulations resulted in macroporous monoliths (pore diameter >500 microm), the majority of the polyHIPEs studied were rigid, microporous monoliths with average pore diameters in the range 10-300 microm. Gravimetric analysis confirmed the porosity of the microporous monoliths as 80-89% with most scaffolds above 84%. These studies demonstrate that emulsion templating can be used to generate rigid, biodegradable scaffolds with highly interconnected pores suitable for tissue engineering scaffolds.     

Cell–scaffold mechanical interplay within engineered tissue

Effective tissue engineering requires appropriate selection of cells and scaffold, where the latter serves as a mechanical and biological support for cell growth and functionality. The optimal combination of cell source and scaffold properties can vary for each desired application. Such preconditions necessitate enhanced understanding of the interactions between cells and scaffold within engineered tissue. Several studies have examined the deforming effects cells induce in scaffolds via exertion of contractile forces. In contrast, other studies focus on the scaffold's biochemical and mechanical properties and their effects on cell behavior.This review summarizes the mechanical interplay between cells and scaffold within engineered tissue. We present evidence for contractile forces exerted by cells on three-dimensional (3D) scaffolds and discuss existing methods for their quantification. In addition, we address some theories related to the effects of scaffold stiffness and mechanical stimulation on cell behavior. Further understanding of the reciprocal effects between cells and scaffold will provide both enhanced knowledge regarding the expected properties of engineered tissue and more competent tissue regeneration techniques.     

A Comparative Study of Fibroblast Behaviors under Cyclic Stress Stimulus and Static Culture on 3D Patterned Matrix

Mechanical stress and patterned surface of the scaffolds has been recognized as a crucial factor in determining cell functionality and tissue development, which in turn can direct the cell responses. In this study, fibroblasts M-3T3 in three-dimensional (3D) honeycomb patterning Chitosan/Poly(L-Lactic Acid) (CS/PLLA) composites was stimulated by a 15% sinusoidal (1 Hz) strain applied by a biodynamic test instrument. The effects of mechanical stimulus on the cell proliferation and basic Fibroblast Growth Factor (bFGF) secretion were studied in comparison to the non-strain groups and blank control. Results show that fibroblasts are able to sense the mechanical stimulation and respond, resulting in a time dependent increase of bFGF secretion and promoting cell proliferation. Moreover, the cells seeded in the scaffolds showed a higher cell proliferation and bFGF secretion. These findings support the hypothesis that suitable mechanical stimulus has positive effect on fibroblasts, and such a 3D honeycomb patterned scaffold may play a positive role in regulating cell behaviors in vitro.     

Cellular infiltration on nanofibrous scaffolds using a modified electrospinning technique

Electrospinning is currently used to fabricate nanofibrous scaffolds for tissue engineering applications. The major problem of these scaffolds is their intrinsically two-dimensional nature which inhibits cellular migration and in-growth. In this study, we have introduced a modified setup of electrospinning to produce three-dimensional nanofibrous scaffolds which allows improved infiltration of cells. An array of focused halogen light bulbs was used to localize the heat in the path of electrospun jet near the collector. The fabricated mats were then seeded with cells in order to evaluate migration and infiltration. After 14 days of culture, a homogenous distribution of cells was observed throughout the scaffolds and showed the three-dimensional architecture of nanofibrous mats. By this novel and simple setup, the prepared electrospun mats will allow the seeded cells to obtain a three-dimensional arrangement which is ideal for tissue engineering applications.     

Current strategies for cell delivery in cartilage and bone regeneration

Several cell-based tissue-engineering therapies are emerging to regenerate damaged tissues. These strategies use autologous cells in combination with bioresorbable delivery materials. Major functions of a delivery scaffold are to provide initial mechanical stability, homogenous three-dimensional cell distribution, improved tissue differentiation, suitable handling and properties for delivery and fixation into patients. Delivery of cells can be achieved using injectable matrices, soft scaffolds, membranes, solid load-bearing scaffolds or immunoprotective macroencapsulation. Thus, to expand the clinical potential, next generation therapies will depend on smart delivery concepts that make use of the regenerative potential of stem cells, morphogenetic growth factors and biomimetic materials.     

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage

The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric alpha-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects.     

Three-dimensional hierarchical composite scaffolds consisting of polycaprolactone, β-tricalcium phosphate, and collagen nanofibers: fabrication, physical properties, and in vitro cell activity for bone tissue regeneration

β-Tricalcium phosphate (β-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to β-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/β-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the β-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the β-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/β-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/β-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and β-TCP particles in the scaffold provide good synergistic effects for cell activity.     

Glycosaminoglycans as regulators of stem cell differentiation

ES (embryonic stem) cell differentiation is dependent on the presence of HS (heparan sulfate). We have demonstrated that, during differentiation, the evolution of specific cell lineages is associated with particular patterns of GAG (glycosaminoglycan) expression. For example, different HS epitopes are synthesized during neural or mesodermal lineage formation. Cell lines mutant for various components of the HS biosynthetic pathway are selectively impaired in their differentiation, with lineage-specific effects observed for some lines. We have also observed that the addition of soluble GAG saccharides to cells, with or without cell-surface HS, can influence the pace and outcome of differentiation, again highlighting specific pattern requirements for particular lineages. We are combining this work with ongoing studies into the design of artificial cell environments where we have optimized three-dimensional scaffolds, generated by electrospinning or by the formation of hydrogels, for the culture of ES cells. By permeating these scaffolds with defined GAG oligosaccharides, we intend to control the mechanical environment of the cells (via the scaffold architecture) as well as their biological signalling environment (using the oligosaccharides). We predict that this will allow us to control ES cell pluripotency and differentiation in a three-dimensional setting, allowing the generation of differentiated cell types for use in drug discovery/testing or in therapeutics.     

Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2

Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a potential alterative cell source for cartilage tissue engineering applications.