Soft Tissue Engineering Research Articles

In vitro evaluation of cytomechanics of osteoblast and fibroblast cell line for enhanced peri‐implant bone and soft tissue engineering

In vitro evaluation of cytomechanics of osteoblast and fibroblast cell line for enhanced peri‐implant bone and soft tissue engineering

Hepatocyte culture on 3D porous scaffolds of PCL/PMCL.

The development of three-dimensional (3D) porous scaffolds for soft tissue engineering mainly focused on manipulation of scaffold properties with cell behaviors. By emulsion freeze-drying method, four types of porous scaffolds were prepared from amorphous poly(4-methy-ε-caprolactone) (PMCL) and semi-crystalline poly(ε-caprolactone) (PCL) at different weight ratios, named as PMCL0, PMCL30, PMCL50 and PMCL70, respectively. Visual observation on cross-sectional images of the scaffolds appeared as sponge-like materials with three-dimensional and highly porous morphologies. However, the pore size, porosity and wettability of blends were not decreased linearly with increasing amorphous PMCL. Distinguished from PMCL30 or PMCL70, PMCL50 preserved intact PCL crystals distributed in amorphous matrix, resulting in the lowest Young's modulus (E) and relatively high wettability. From in vitro cell culture, it was observed that PMCL50 scaffold supported human induced hepatocytes (hiHeps) proliferation and function preservation best among all scaffolds. hiHeps on PMCL50 inclined to adopt fibroblastic morphology, whereas formed spheroidal morphology on PMCL0. It was suggested that our bare scaffolds with tailored properties have shown remarkable capability towards hiHep proliferation and function expression.

Synthetic polymer scaffolds for soft tissue engineering.

Tissue engineering (TE) and regenerative medicine are progressively developed areas due to many novel tissue replacements and implementation strategies. Increasing knowledge involving the fabrication of biomaterials with advanced physicochemical and biological characteristics, successful isolation and preparation of stem cells, incorporation of growth and differentiation factors, and biomimetic environments gives us a unique opportunity to develop various types of scaffolds for TE. The current strategies for soft tissue reconstitution or regeneration highlight the importance of novel regenerative therapies in cases of significant soft tissue loss and in cases of congenital defects, disease, trauma and ageing. Various types of biomaterials and scaffolds have been tested for soft tissue regeneration. The synthetic types of materials have gained great attention due to high versatility, tunability and easy functionalization for better biocompatibility. This article reviews the current materials that are usually the most used for the fabrication of scaffolds for soft TE; in addition, the types of scaffolds together with examples of their applications for the regenerative purposes of soft tissue, as well as their major physicochemical characteristics regarding the increased applicability of these materials in medicine, are reviewed.

Advances in the synthesis of non-isocyanate polyurethanes

Polyurethane (PU) is a most commonly used polymer for various application such as rigid and flexible foams, coatings etc. Also another class known as biodegradable PUs are used in specialized medical fields like soft-tissue engineering, regenerative medicine and also drug delivery systems, However, the degradation of the polyurethanes derived traditionally from general diisocyanates like 4,40-methylene diphenyl diisocyanate and toluene diisocyanate gives toxic products. Therefore, it is imperative to find an alternative route for the synthesis of PU which involves least or no toxic reagents. The consequently formed products are named non-isocyanate polyurethanes (NIPU). Recently, few alternative synthesis methods have been suggested by many researchers like a synthesis of polyhydroxyurethanes by polyaddition of diamines with cyclic carbonates. Such methods use functionalized vegetable oil as a biocompatible monomer which has led to a new range of biodegradable, renewable and sustainable source for NIPUs. Use of cyclocarbonates class of compounds has shown potential in the preparation of green PUs. Self-polycondensation reactions between AB-type fatty monomers bearing acyl azide and hydroxyl functionalities will take part. Here, the isocyanate group formation takes place in situ via Curtius rearrangement. The only disadvantage of this method is that the acyl azide groups are obtained by the harmful reaction of sodium azide with carboxylic acids. Bio-renewable sources represent a rich source of precursors for the synthesis of polyols and polyisocyanates. Many such sustainable routes for the synthesis of NIPUs reported in the literature are presented in the following article.

Mechanically-enhanced polysaccharide-based scaffolds for tissue engineering of soft tissues

Collagen-based materials are probably among the most used class of biomaterials in tissue engineering and regenerative medicine. Although collagen is often privileged for providing a suitable substrate on which cells can be cultured or a matrix in which cells can be dispersed, its mechanical properties represent a major limitation for clinical translation and even for handling of the obtained regenerated tissue. In this work, the combination of polysaccharides chitosan (Ch) and xanthan gum (X) was investigated as an alternative for scaffolds for soft tissue engineering. Moreover, in an attempt to reach a compromise between obtaining highly porous biomaterials while maintaining appropriate mechanical properties, a surfactant (Kolliphor® P188, K) was added to Ch-X matrices to generate pores, while silicone rubber (Silpuran® 2130A/B, S) was used to balance their mechanical properties. Addition of K (10 or 25% w/w) increased the porosity and pore-dimensions, while addition of S improved by up to 156% and 85% the elastic moduli and the elastic behavior of Ch-X-based scaffolds, under both compressive and tensile loads, respectively, at 50% strain. Relaxation tests confirmed that these materials do have a viscoelastic behavior. The presence of S increased thickness and microscale surface roughness and did not affect liquid uptake and stability, thrombogenicity, biodegradation and cytotoxicity of polysaccharide-based scaffolds. In conclusion, this work shows that Ch-X-S porous blends constitute suitable scaffolds for soft tissue engineering.

Focal Adhesion Kinase and ROCK Signaling Are Switch-Like Regulators of Human Adipose Stem Cell Differentiation towards Osteogenic and Adipogenic Lineages.

Adipose tissue is an attractive stem cell source for soft and bone tissue engineering applications and stem cell therapies. The adipose-derived stromal/stem cells (ASCs) have a multilineage differentiation capacity that is regulated through extracellular signals. The cellular events related to cell adhesion and cytoskeleton have been suggested as central regulators of differentiation fate decision. However, the detailed knowledge of these molecular mechanisms in human ASCs remains limited. This study examined the significance of focal adhesion kinase (FAK), Rho-Rho-associated protein kinase (Rho-ROCK), and their downstream target extracellular signal-regulated kinase 1/2 (ERK1/2) on hASCs differentiation towards osteoblasts and adipocytes. Analyses of osteogenic markers RUNX2A, alkaline phosphatase, and matrix mineralization revealed an essential role of active FAK, ROCK, and ERK1/2 signaling for the osteogenesis of hASCs. Inhibition of these kinases with specific small molecule inhibitors diminished osteogenesis, while inhibition of FAK and ROCK activity led to elevation of adipogenic marker genes AP2 and LEP and lipid accumulation implicating adipogenesis. This denotes to a switch-like function of FAK and ROCK signaling in the osteogenic and adipogenic fates of hASCs. On the contrary, inhibition of ERK1/2 kinase activity deceased adipogenic differentiation, indicating that activation of ERK signaling is required for both adipogenic and osteogenic potential. Our findings highlight the reciprocal role of cell adhesion mechanisms and actin dynamics in regulation of hASC lineage commitment. This study enhances the knowledge of molecular mechanisms dictating hASC differentiation and thus opens possibilities for more efficient control of hASC differentiation.

Hydrogel from Acellular Porcine Adipose Tissue Accelerates Wound Healing by Inducing Intradermal Adipocyte Regeneration

As an important component of the skin, intradermal adipocytes are closely associated with skin homeostasis and wound healing. Although studies have focused on the role of fibroblasts, keratinocytes, and inflammatory cells in wound healing, the role of adipocytes has not been fully investigated. Here, we verified whether the induction of adipocyte regeneration in a wound bed can effectively promote wound healing, finding that the hydrogel from acellular porcine adipose tissue in combination with adipose-derived stem cells can induce in situ adipogenesis in the wound microenvironment. The newly regenerated adipocytes enhanced fibroblast migration, accelerated wound closing, and enhanced wound epithelialization. More importantly, newly formed intact skin structure was observed after treating the wound with adipose-derived stem cell-loaded hydrogel from acellular porcine adipose tissue. These results show that hydrogel from acellular porcine adipose tissue might substantially improve re-epithelialization, angiogenesis, and skin-appendage regeneration, making it a promising therapeutic biomaterial for skin wound healing.

Nanocomposite smart hydrogels with improved responsiveness and mechanical properties: A mini review

ABSTRACTResponsive hydrogels have the ability to change their volume, transparency, or other properties in response to external chemical and/or physical stimuli. The responsiveness properties including responsive rate and degree, as well as mechanical properties such as Young's modulus, toughness, breaking strength, and breaking strain are crucial parameters of the smart hydrogels that determine the scope of hydrogel applications such as soft actuators, artificial muscles, and tissue engineering scaffolds. In this paper, the development of the nanocomposite smart hydrogels, which can achieve both improved responsiveness and mechanical properties, is reviewed. First, the fabrication approaches for building the nanocomposite networks by doping organic or inorganic nanomaterials via crosslinking or blending strategies are introduced. Then, the mechanisms used to improve both responsiveness and mechanical properties of nanocomposite responsive hydrogels are discussed. Finally, the perspectives as well as current challenges of such nanocomposite responsive hydrogels are addressed. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018, 56, 1306–1313

Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits

Entubulating devices to repair peripheral nerve injuries are limited in their effectiveness particularly for critical gap injuries. Current clinically used nerve guidance conduits are often simple tubes, far stiffer than that of the native tissue. This study assesses the use of poly(glycerol sebacate methacrylate) (PGSm), a photocurable formulation of the soft biodegradable material, PGS, for peripheral nerve repair. The material was synthesized, the degradation rate and mechanical properties of material were assessed and nerve guidance conduits were structured via stereolithography. In vitro cell studies confirmed PGSm as a supporting substrate for both neuronal and glial cell growth. Ex vivo studies highlight the ability of the cells from a dissociated dorsal root ganglion to grow out and align along the internal topographical grooves of printed nerve guide conduits. In vivo results in a mouse common fibular nerve injury model show regeneration of axons through the PGSm conduit into the distal stump after 21 days. After conduit repair levels of spinal cord glial activation (an indicator for neuropathic pain development) were equivalent to those seen following graft repair. In conclusion, results indicate that PGSm can be structured via additive manufacturing into functional NGCs. This study opens the route of personalized conduit manufacture for nerve injury repair. Statement of SignificanceThis study describes the use of photocurable of Poly(Glycerol Sebacate) (PGS) for light-based additive manufacturing of Nerve Guidance Conduits (NGCs). PGS is a promising flexible biomaterial for soft tissue engineering, and in particular for nerve repair. Its mechanical properties and degradation rate are within the desirable range for use in neuronal applications. The nerve regeneration supported by the PGS NGCs is similar to an autologous nerve transplant, the current gold standard. A second assessment of regeneration is the activation of glial cells within the spinal cord of the tested animals which reveals no significant increase in neuropathic pain by using the NGCs. This study highlights the successful use of a biodegradable additive manufactured NGC for peripheral nerve repair.

Use of Gold Nanoparticles to Investigate the Drug Embedding and Releasing Performance in Biodegradable Poly(glycerol sebacate)

Poly(glycerol sebacate) (PGS) is a soft elastomer with excellent biocompatibility, and it has great potential in the application of soft tissue engineering and drug delivery. However, there is stil...

Skin Regeneration with a Scaffold of Predefined Shape and Bioactive Peptide Hydrogels.

We developed a highly elastic customized scaffold for soft tissue regeneration and combined them with bioactive hydrogels with stem cell-inducing ability. This was done to mimic mechanical properties of native soft tissues and improve the viability of transplanted cells as well as efficiency of tissue regeneration. The proposed study was aimed at evaluating various characteristics of scaffolds and investigating their tissue-regenerating ability. Finger-shaped porous scaffolds were successfully fabricated by an indirect 3D printing of poly (L-lactide-co-ɛ-caprolactone) (PLCL), which provides high elasticity for soft tissue engineering. In addition, a self-assembling peptide hydrogel coupled with substance P (RARADADARARADADA/RARADADARARADADA-substance P, RADA16/RADA16-SP) was used to accelerate angiogenesis and recruit intrinsic mesenchymal stem cells (MSCs). This study included three kinds of groups: Group I = PLCL scaffold with human dermal fibroblasts (HDFs) (P+C), Group II = PLCL scaffold with HDFs and RADA16 (P+C+R), and Group III = PLCL scaffold with HDFs, RADA16, and RADA16-SP (P+C+R+S). The samples were implanted into immunodeficient mice subcutaneously and harvested at 1 and 4 weeks. Tissue regeneration was evaluated by histological analysis with hematoxylin and eosin (H&E) and Masson's trichrome (MT) staining. The images showed that a large number of cells were recruited into the scaffolds, and collagen was deposited in the constructs of the P+C+R+S group. Additionally, recruitment of MSCs, angiogenesis, and collagen were observed by immunofluorescence staining. The results show that the P+C+R+S group had more type I and type III collagen, which are formed in soft tissues, and were deposited on the scaffold compared with the other groups. Moreover, more blood vessels and MSCs were induced in the P+C+R+S group than in those of the P + C and P+C+R groups. Consequently, the results suggest that the construct of the customized porous PLCL scaffold and RADA16/RADA16-SP hydrogel could be a good treatment modality to treat skin defects.

Labeling of Collagen Type I Templates with a Naturally Derived Contrast Agent for Noninvasive MR Imaging in Soft Tissue Engineering.

In vivo monitoring of tissue-engineered constructs is important to assess their integrity, remodeling, and degradation. However, this is challenging when the contrast with neighboring tissues is low, necessitating labeling with contrast agents (CAs), but current CAs have limitations (i.e., toxicity, negative contrast, label instability, and/or inappropriate size). Therefore, a naturally derived hemin-L-lysine (HL) complex is used as a potential CA to label collagen-based templates for magnetic resonance imaging (MRI). Labeling does not change the basic characteristics of the collagen templates. When hybrid templates composed of collagen type I reinforced with degradable polymers are subcutaneously implanted in mice, longitudinal visualization by MRI is possible with good contrast and in correlation with template remodeling. In contrast, unlabeled collagen templates are hardly detectable and the fate of these templates cannot be monitored by MRI. Interestingly, tissue remodeling and vascularization are enhanced within HL-labeled templates. Thus, HL labeling is presented as a promising universal imaging marker to label tissue-engineered implants for MRI, which additionally seems to accelerate tissue regeneration.

High-Performance Photopolymerized Poly(vinyl alcohol)/Silica Nanocomposite Hydrogels with Enhanced Cell Adhesion.

Poly(vinyl alcohol) (PVA) hydrogels have been considered as promising implants for various soft tissue engineering applications because of their tissue-like viscoelasticity and biocompatibility. However, two critical barriers including lack of sufficient mechanical properties and non-tissue-adhesive characterization limit their application as tissue substitutes. Herein, PVA is methacrylated with ultralow degrees of substitution of methacryloyl groups to produce PVA-glycidyl methacrylate (GMA). Subsequently, the PVA-GMA/methacrylate-functionalized silica nanoparticle (MSi)-based nanocomposite hydrogels are developed via the photopolymerization approach. Interestingly, both PVA-GMA-based hydrogels and PVA-GMA/MSi-based nanocomposite hydrogels exhibit outstanding compressive properties, which cannot be damaged through compressive stress-strain tests in the allowable scope of a tensile tester. Moreover, PVA-GMA/MSi-based nanocomposite hydrogels demonstrate excellent tensile properties compared with neat PVA-GMA-based hydrogels, and 15-, 14-, and 24-fold increase in fracture stress, elastic modulus, and toughness, respectively, is achieved for the PVA-GMA/MSi-based hydrogels with 10 wt % of MSi. These remarkable enhancements can be ascribed to the amount of long and flexible polymer chains of PVA-GMA and the strong interactions between the MSi and PVA-GMA chains. More interestingly, exciting improvements in the cell adhesion can also be successfully achieved by the incorporation of MSi nanoparticles.

BSA Adsorption on Porous Scaffolds Prepared from BioPEGylated Poly(3-Hydroxybutyrate)

Porous scaffolds for tissue engineering have been prepared from poly(3-hydroxybutyrate) (PHB) and a copolymer of poly(3-hydroxybutyrate) and polyethylene glycol (PHB-PEG) produced by bioPEGylation. The morphology of the scaffolds and their capacity for adsorption of the model protein bovine serum albumin (BSA) have been studied. Scaffolds produced from bioPEGylated PHB adsorbed more BSA, whereas the share of protein irreversibly adsorbed on these scaffolds was significantly lower (33%) than in the case of PHB homopolymer-based scaffolds (47%). The effect of protein adsorption on scaffold biocompatibility in vitro was tested in an experiment that involved the cultivation of fibroblasts (line COS-1) on the scaffolds. PHB-PEG scaffolds had a higher capacity for supporting cell growth than PHB-based scaffolds. Thus, the bioPEGylated PHB-based polymer scaffolds developed in the present study have considerable potential for use in soft tissue engineering.

Interfacial tissue engineering of heart regenerative medicine based on soft cell-porous scaffolds.

Myocardial infarction (MI), occurs when the coronary artery is occluded resulting in the hypoxia of areas in heart tissue, is increasing in recent years because of the population ageing and lifestyle changes. Currently, there is no ideal therapeutic scheme because of the limitation of MI therapeutic strategies due to the lack of regenerative ability of the heart cells in adult humans. Recent advances in tissue engineering and regenerative medicine brings hope to the MI therapy and current studies are focusing on restoring the function and structure of damaged tissue by delivering exogenous cells or stimulating endogenous heart cells. However, attempts to directly inject stem cells or cardiomyocytes to the infract zone often lead to rapid cell death and abundant cell loss. To address this challenge, various soft repair cells and porous scaffold materials have been integrated to improve cell retention and engraftment and preventing left ventricle (LV) dilatation. In this article, we will review the current method for heart regeneration based on soft cell-porous scaffold interfacial tissue engineering including common stem cell types, biomaterials, and cardiac patch and will discuss potential future directions in this area.

Development of Biomimetic Hybrid Porous Scaffold of Chitosan/Polyvinyl Alcohol/Carboxymethyl Cellulose by Freeze-Dried and Salt Leached Technique.

Freeze drying and salt leaching methods were applied to fabricate Chitosan/Poly(vinyl alcohol)/Carboxymethyl cellulose (CPCMC) biomimetic porous scaffolds for soft tissue engineering. The properties of these scaffolds were investigated and compared to those by freeze drying and salt leaching methods respectively. The salt-leached CS/PVA/CMC scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size. The mechanical strength of the scaffolds increased with the porosity, and were easily modulated by the addition of carboxymethyl cellulose. The morphology of the porous scaffolds observed using a SEM exhibited good porosity and interconnectivity of pores. MTT assay using L929 fibroblast cells demonstrated that the cell viability of the porous scaffold was good. Scaffolds prepared by salt leached method show larger swelling capacity, and mechanical strength, potent antibacterial activity and more cell viability than freeze dried method. It is found that salt leaching method has distinguished characteristics of simple, efficient, feasible and less economic than freeze dried scaffolds.

The quest for mechanically and biologically functional soft biomaterials via soft network composites.

Developing multifunctional soft biomaterials capable of addressing all the requirements of the complex tissue regeneration process is a multifaceted problem. In order to tackle the current challenges, recent research efforts are increasingly being directed towards biomimetic design concepts that can be translated into soft biomaterials via advanced manufacturing technologies. Among those, soft network composites consisting of a continuous hydrogel matrix and a reinforcing fibrous network closely resemble native soft biological materials in terms of design and composition as well as physicochemical properties. This article reviews soft network composite systems with a particular emphasis on the design, biomaterial and fabrication aspects within the context of soft tissue engineering and drug delivery applications.

Modelling of silk-reinforced PDMS properties for soft tissue engineering applications.

Polydimethylsiloxane (PDMS) is widely used in biomedical research and technology, but its mechanical properties should be tuned according to the desired product specifications. Mixing ratio of base polymer to curing agent or additives enables its mechanical properties to be manipulated and fit to mechanical properties of biological tissues. In this paper, we analysed the effect of mechanical load on silk-reinforced PDMS depending on silk concentration. We prepared cylinder-type PDMS samples with different silk concentrations and performed cyclic uniaxial compression tests with a fixed magnitude of applied strain. Next, we analysed the mechanical charascteristics of PDMS using computational modelling. The stress-strain data within the large-strain region of different PDMS cylinders without silk and with 1%, 5% and 10% silk concentrations was fitted to non-linear second order Mooney-Rivlin, and third-order Ogden models. The results show the equivalence of both models for investigated strain region of PDMS. On the other hand, PDMS cylinders with 10% silk concentration allowed the successful fitting of experimental data just for the second-order Mooney-Rivlin model, while all numerical probes to find an appropriate fitting parameters for third-order Ogden models were unsuccessful. The second-order Mooney-Rivlin model is preferable for analysing the properties of silk-reinforced PDMS over the entire measurement range.

Self-Assembly of a Dentinogenic Peptide Hydrogel.

Current standard of care for treating infected dental pulp, root canal therapy, retains the physical properties of the tooth to a large extent, but does not aim to rejuvenate the pulp tissue. Tissue-engineered acellular biomimetic hydrogels have great potential to facilitate the regeneration of the tissue through the recruitment of autologous stem cells. We propose the use of a dentinogenic peptide that self-assembles into β-sheet-based nanofibers that constitute a biodegradable and injectable hydrogel for support of dental pulp stem cells. The peptide backbone contains a β-sheet-forming segment and a matrix extracellular phosphoglycoprotein mimic sequence at the C-terminus. The high epitope presentation of the functional moiety in the self-assembled nanofibers may enable recapitulation of a functional niche for the survival and proliferation of autologous cells. We elucidated the hierarchical self-assembly of the peptide through biophysical techniques, including scanning electron microscopy and atomic force microscopy. The material property of the self-assembled hydrogel was probed though oscillatory rheometry, demonstrating its thixotropic nature. We also demonstrate the cytocompatibility of the hydrogel with respect to fibroblasts and dental pulp stem cells. The self-assembled peptide platform holds promise for guided dentinogenesis and it can be tailored to a variety of applications in soft tissue engineering and translational medicine in the future.

Electrospinning Process and Structure Relationship of Biobased Poly(butylene succinate) for Nanoporous Fibers.

Biobased poly(butylene succinate) (BioPBS) was electrospun to create hierarchical, highly porous fibers. Various grades of BioPBS were dissolved in one of the three solutions: chloroform, a co-solvent system of chloroform/N,N-dimethylformamide (DMF), or chloroform/dimethyl sulfoxide (DMSO). These solutions were then electrospun at room temperature to produce nanoporous micron-sized fibers. The variables investigated were the solvent system used, grade of BioPBS, concentration of BioPBS, applied voltage, and the distance between the electrodes. In determining the optimal solution and electrospinning conditions, it was found that solution properties such as the solvent system, the grade of BioPBS, and the concentration of BioPBS had a significant effect on the fiber morphology. A chloroform/DMSO cosolvent system resulted in less bead defects among fibers compared to those produced from chloroform/DMF systems, regardless of the BioPBS grade. An increase in BioPBS concentration resulted in the reduction of bead defects, which at 15 (% w/v) resulted in bead-free uniform fibers. Increasing BioPBS concentration also increased the porosity of the fibers while reducing the pore size. Dynamic mechanical analysis showed that the reduction of bead defects resulted in increased tensile strength and Young’s modulus of the electrospun fibrous nonwoven mat. The results of this study show that electrospun BioPBS fibers have high porosity at the micro- and nanoscale, resulting in a hierarchical structure that has sufficient mechanical properties for potential applications in wound healing and soft tissue engineering.