Cellular Response of Limbal Stem Cells on Poly (Hydroxybuthyrate-co-Hydroxyvalerate) Porous Scaffolds for Ocular Surface Bioengineering

The aim of this study was to develop a modified-porous poly (3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffold for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human limbal stem cell was used to evaluate the biocompatibility of substrates (porous scaffold, human amniotic membrane and thermoresponsive substrate) based on their viability, proliferation, and attachment ability. Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the scaffold surface. Microscopic figures showed that the human limbal stem cell was firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the porous scaffold and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Gene expression results revealed no change in the expression profile of LECs grown on scaffold when compared to those grown on human amniotic membrane or thermo responsive substrate. In addition, porous PHBV substrate provides not only a milieu supporting LSCs expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.     

Fibrin-Plasma Rich in Growth Factors Membrane for the Treatment of a Rabbit Alkali-Burn Lesion

The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.     

Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role.     

Partial Decellularized Scaffold Combined with Autologous Nasal Epithelial Cell Sheet for Tracheal Tissue Engineering

Decellularization has emerged as a potential solution for tracheal replacement. As a fully decellularized graft failed to achieve its purposes, the de-epithelialization partial decellularization protocol appeared to be a promising approach for fabricating scaffolds with preserved mechanical properties and few immune rejection responses after transplantation. Nevertheless, a lack of appropriate concurrent epithelialization treatment can lead to luminal stenosis of the transplant and impede its eventual success. To improve re-epithelialization, autologous nasal epithelial cell sheets generated by our cell sheet engineering platform were utilized in this study under an in vivo rabbit model. The newly created cell sheets have an intact and transplantable appearance, with their specific characteristics of airway epithelial origin being highly expressed upon histological and immunohistochemical analysis. Subsequently, those cell sheets were incorporated with a partially decellularized tracheal graft for autograft transplantation under tracheal partial resection models. The preliminary results two months post operation demonstrated that the transplanted patches appeared to be wholly integrated into the host trachea with adequate healing of the luminal surface, which was confirmed via endoscopic and histologic evaluations. The satisfactory result of this hybrid scaffold protocol could serve as a potential solution for tracheal reconstructions in the future.     

Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery

Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits) and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH₄OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation.     

Cellular Response of Limbal Stem Cells on PHBV/Gelatin Nanofibrous Scaffold for Ocular Epithelial Regeneration

The aim of this study was to develop a blend of nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/gelatin substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human Limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold, and human amniotic membrane) based on their phenotypic profile, viability, proliferation, and attachment ability. Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time–PCR results revealed no change in the expression profile of LSCs grown on nanofibrous substrate when compared to those grown on human amniotic membrane. In addition, electrospun nanofibrous PHBV substrate provides not only a milieu supporting LSCs expansion, but also serves as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.     

Cartilage Formation In Vivo Using High Concentration Collagen-Based Bioink with MSC and Decellularized ECM Granules

The aim of this study was to verify the applicability of high-concentration collagen-based bioink with MSC (ADSC) and decellularized ECM granules for the formation of cartilage tissue de novo after subcutaneous implantation of the scaffolds in rats. The printability of the bioink (4% collagen, 2.5% decellularized ECM granules, derived via 280 μm sieve) was shown. Three collagen-based compositions were studied: (1) with ECM; (2) with MSC; (3) with ECM and MSC. It has been established that decellularized ECM granules are able to stimulate chondrogenesis both in cell-free and MSC-laden scaffolds. Undesirable effects have been identified: bone formation as well as cartilage formation outside of the scaffold area. The key perspectives and limitations of ECM granules (powder) application have been discussed.     

Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct thick tissues because of the time required for sufficient vascular network formation. Recently, the co-culture of graft tissue with vascular cells before transplantation has attracted attention as a way of promoting capillary angiogenesis. Although the co-cultured vascular cells can directly contribute to blood vessel formation within the tissue, a key objective that needs to be met is the construction of a continuous circulatory structure. Previously described strategies to reconstruct blood vessels include the culture of endothelial cells in a scaffold that contains microchannels or within the original vascular framework after decellularization of an entire organ. The technique, as developed by authors, involves the progressive stacking of three-layered cell sheets onto a vascular bed to induce the formation of a capillary network within the cell sheets. This approach enables the construction of thick, functional tissue of high cell density that can be transplanted by anastomosing its artery and vein (provided by the vascular bed) with host blood vessels.     

The Regeneration of Large-Sized and Vascularized Adipose Tissue Using a Tailored Elastic Scaffold and dECM Hydrogels

A dome-shaped elastic poly(l-lactide-co-caprolactone) (PLCL) scaffold with a channel and pore structure was fabricated by a combinative method of 3D printing technology and the gel pressing method (13 mm in diameter and 6.5 mm in thickness) for patient-specific regeneration. The PLCL scaffold was combined with adipose decellularized extracellular matrix (adECM) and heart decellularized extracellular matrix (hdECM) hydrogels and human adipose-derived stem cells (hADSCs) to promote adipogenesis and angiogenesis. These scaffolds had mechanical properties similar to those of native adipose tissue for improved tissue regeneration. The results of the in vitro real-time PCR showed that the dECM hydrogel mixture induces adipogenesis. In addition, the in vivo study at 12 weeks demonstrated that the tissue-engineered PLCL scaffolds containing the hydrogel mixture (hdECM/adECM (80:20)) and hADSCs promoted angiogenesis and adipose tissue formation, and suppressed apoptosis. Therefore, we expect that our constructs will be clinically applicable as material for the regeneration of patient-specific large-sized adipose tissue.     

Fabrication of 3D melt electrowritting multiphasic scaffold with bioactive and osteoconductivite functionalities for periodontal regeneration

This work aimed to design a multifunctional biphasic 3D scaffold for periodontal tissue regeneration. A 3D fibrous scaffold made from medical-grade poly (ε-caprolactone) (PCL) with high porosity (>90%) and well-oriented fibres was fabricated by a custom design melt electrowriting (MEW) device. A biomimetic process was employed to form a bioactive calcium phosphate (CaP) layer with nanostructure (nanoflakes-like) morphology onto the 3D MEW fibrous surface to stimulate rapid bone formation. Primary human osteoblasts (hOBs) were seeded within the coated 3D fibrous scaffolds for 28 days to acquire the bone compartment of the tissue-engineered construct (TEC). The biphasic construct was obtained by placing an established in vitro periodontal ligament (PDL) cell sheet onto the surface of the bone compartment. Subsequently, a decellularized multiphasic TEC by exploiting a lyophilization approach was obtained. Laser scanning confocal microscopy and scanning electron microscopy confirmed the retention of a functional extracellular matrix within the PDL and bone compartments following scaffold decellularization and lyophilization processes. These findings suggest that lyophilized decellularized biphasic 3D constructs with high porosity constitute a viable ‘off the shelf’ strategy for developing an extracellular matrix-based product to facilitate periodontal regeneration.     

Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.     

Albumin-based biomaterial for lung tissue engineering applications

The role of albumin-based biomaterials in tissue engineering (TE) cannot be overemphasized. The authors review the role of albumin in lungs scaffold grafting, which promotes cell seeding. Albumin grafted on decellularized lungs scaffold is presented as a great support material for cell-tissue interaction as well as for ease in attachment, growth, and differentiation when seeded with different types of cells. Albumin scaffold fabrication from different sources is a promising approach that may facilitate medical treatments from bench-to-bed, although the role of this scaffold in lungs surfactant proteins regeneration and binding needs to be fully elucidated.     

Distal Lung Microenvironment Triggers Release of Mediators Recognized as Potential Systemic Biomarkers for Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model’s applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.     

BIOMIMETIC GRADIENT HYDROGELS FOR TISSUE ENGINEERING

During tissue morphogenesis and homeostasis, cells experience various signals in their environments, including gradients of physical and chemical cues. Spatial and temporal gradients regulate various cell behaviours such as proliferation, migration, and differentiation during development, inflammation, wound healing, and cancer. One of the goals of functional tissue engineering is to create microenvironments that mimic the cellular and tissue complexity found in vivo by incorporating physical, chemical, temporal, and spatial gradients within engineered three-dimensional (3D) scaffolds. Hydrogels are ideal materials for 3D tissue scaffolds that mimic the extracellular matrix (ECM). Various techniques from material science, microscale engineering, and microfluidics are used to synthesise biomimetic hydrogels with encapsulated cells and tailored microenvironments. In particular, a host of methods exist to incorporate micrometer to centimetre scale chemical and physical gradients within hydrogels to mimic the cellular cues found in vivo. In this review, we draw on specific biological examples to motivate hydrogel gradients as tools for studying cell-material interactions. We provide a brief overview of techniques to generate gradient hydrogels and showcase their use to study particular cell behaviours in two-dimensional (2D) and 3D environments. We conclude by summarizing the current and future trends in gradient hydrogels and cell-material interactions in context with the long-term goals of tissue engineering.     

The incorporation and controlled release of platelet‐rich plasma‐derived biomolecules from polymeric tissue engineering scaffolds

Platelet‐rich plasma (PRP) has been gaining popularity in recent years as a cost‐effective material capable of stimulating healing in a number of different clinical applications. As the clinical role of PRP has been growing so too has its prevalence in the fields of tissue engineering and regenerative medicine, particularly in the field of extracellular matrix (ECM) analogue scaffold fabrication. As polymeric scaffold fabrication techniques strive to create structures that ever more closely replicate the native ECM's form and function, the need for increased scaffold bioactivity becomes more pronounced. PRP, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors and cytokines capable of stimulating cellular activity through enhanced chemotaxis, proliferation and ECM production. The ability to incorporate such a potent bioactive milieu into a polymeric tissue engineering scaffold, which lacks intrinsic cell signaling molecules, may help to promote scaffold integration with native tissues and increase the overall patency of polymeric ECM analogue structures. This mini‐review briefly discusses the physiological basis of PRP and its current clinical use, as well as the potential role that PRP may play in the future of polymeric tissue engineering scaffold design. Copyright © 2012 Society of Chemical Industry     

Hyaluronic Acid and a Short Peptide Improve the Performance of a PCL Electrospun Fibrous Scaffold Designed for Bone Tissue Engineering Applications

Bone tissue engineering is a rapidly developing, minimally invasive technique for regenerating lost bone with the aid of biomaterial scaffolds that mimic the structure and function of the extracellular matrix (ECM). Recently, scaffolds made of electrospun fibers have aroused interest due to their similarity to the ECM, and high porosity. Hyaluronic acid (HA) is an abundant component of the ECM and an attractive material for use in regenerative medicine; however, its processability by electrospinning is poor, and it must be used in combination with another polymer. Here, we used electrospinning to fabricate a composite scaffold with a core/shell morphology composed of polycaprolactone (PCL) polymer and HA and incorporating a short self-assembling peptide. The peptide includes the arginine-glycine-aspartic acid (RGD) motif and supports cellular attachment based on molecular recognition. Electron microscopy imaging demonstrated that the fibrous network of the scaffold resembles the ECM structure. In vitro biocompatibility assays revealed that MC3T3-E1 preosteoblasts adhered well to the scaffold and proliferated, with significant osteogenic differentiation and calcium mineralization. Our work emphasizes the potential of this multi-component approach by which electrospinning, molecular self-assembly, and molecular recognition motifs are combined, to generate a leading candidate to serve as a scaffold for bone tissue engineering.     

Decreased Lymphangiogenic Activities and Genes Expression of Cord Blood Lymphatic Endothelial Progenitor Cells (VEGFR3+/Pod+/CD11b+ Cells) in Patient with Preeclampsia

The abnormal development or disruption of the lymphatic vasculature has been implicated in metabolic and hypertensive diseases. Recent evidence suggests that the offspring exposed to preeclampsia (PE) in utero are at higher risk of long-term health problems, such as cardiovascular and metabolic diseases in adulthood, owing to in utero fetal programming. We aimed to investigate lymphangiogenic activities in the lymphatic endothelial progenitor cells (LEPCs) of the offspring of PE. Human umbilical cord blood LEPCs from pregnant women with severe PE (n = 10) and gestationally matched normal pregnancies (n = 10) were purified with anti-vascular endothelial growth factor receptor 3 (VEGFR3)/podoplanin/CD11b microbeads using a magnetic cell sorter device. LEPCs from PE displayed significantly delayed differentiation and reduced formation of lymphatic endothelial cell (LEC) colonies compared with the LEPCs from normal pregnancies. LECs differentiated from PE-derived LEPCs exhibited decreased tube formation, migration, proliferation, adhesion, wound healing, and 3D-sprouting activities as well as increased lymphatic permeability through the disorganization of VE-cadherin junctions, compared with the normal pregnancy-derived LECs. In vivo, LEPCs from PE showed significantly reduced lymphatic vessel formation compared to the LEPCs of the normal pregnancy. Gene expression analysis revealed that compared to the normal pregnancy-derived LECs, the PE-derived LECs showed a significant decrease in the expression of pro-lymphangiogenic genes (GREM1, EPHB3, VEGFA, AMOT, THSD7A, ANGPTL4, SEMA5A, FGF2, and GBX2). Collectively, our findings demonstrate, for the first time, that LEPCs from PE have reduced lymphangiogenic activities in vitro and in vivo and show the decreased expression of pro-lymphangiogenic genes. This study opens a new avenue for investigation of the molecular mechanism of LEPC differentiation and lymphangiogenesis in the offspring of PE and subsequently may impact the treatment of long-term health problems such as cardiovascular and metabolic disorders of offspring with abnormal development of lymphatic vasculature.     

Enhanced Regeneration of Vascularized Adipose Tissue with Dual 3D-Printed Elastic Polymer/dECM Hydrogel Complex

A flexible and bioactive scaffold for adipose tissue engineering was fabricated and evaluated by dual nozzle three-dimensional printing. A highly elastic poly (L-lactide-co-ε-caprolactone) (PLCL) copolymer, which acted as the main scaffolding, and human adipose tissue derived decellularized extracellular matrix (dECM) hydrogels were used as the printing inks to form the scaffolds. To prepare the three-dimensional (3D) scaffolds, the PLCL co-polymer was printed with a hot melting extruder system while retaining its physical character, similar to adipose tissue, which is beneficial for regeneration. Moreover, to promote adipogenic differentiation and angiogenesis, adipose tissue-derived dECM was used. To optimize the printability of the hydrogel inks, a mixture of collagen type I and dECM hydrogels was used. Furthermore, we examined the adipose tissue formation and angiogenesis of the PLCL/dECM complex scaffold. From in vivo experiments, it was observed that the matured adipose-like tissue structures were abundant, and the number of matured capillaries was remarkably higher in the hydrogel–PLCL group than in the PLCL-only group. Moreover, a higher expression of M2 macrophages, which are known to be involved in the remodeling and regeneration of tissues, was detected in the hydrogel–PLCL group by immunofluorescence analysis. Based on these results, we suggest that our PLCL/dECM fabricated by a dual 3D printing system will be useful for the treatment of large volume fat tissue regeneration.     

Image-Based Method to Quantify Decellularization of Tissue Sections

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.