Development of heparinized and hepatocyte growth factor-coated acellular scaffolds using porcine carotid arteries.

Tissue-engineered blood vessel substitutes have been developed due to the lack of suitable small-diameter vascular grafts. Xenogeneic extracellular matrix (ECM) scaffolds have the potential to provide an ideal source for off-the-shelf vascular grafts. In this study, porcine carotid arteries were used to develop ECM scaffolds by decellularization and coating with heparin and hepatocyte growth factor (HGF). After decellularization, cellular and nucleic materials were successfully removed with preservation of the main compositions (collagen, elastin, and basement membrane) of the native ECM. The ultimate tensile strength, suture strength, and burst pressure were significantly increased after cross-linking. Pore size distribution analysis revealed a porous structure within ECM scaffolds with a high distribution of pores larger than 10 µm. Heparinized scaffolds exhibited sustained release of heparin in vitro and showed potent anticoagulant activity by prolonging activated partial thromboplastin time. The scaffolds showed an enhanced HGF binding capacity as well as a constant release of HGF as a result of heparin modification. When implanted subcutaneously in rats, the modified scaffolds revealed good biocompatibility with enzyme degradation resistance, mitigated immune response, and anti-calcification. In conclusion, heparinized and HGF-coated acellular porcine carotid arteries may be a promising biological scaffold for tissue-engineered vascular grafts.

Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering.

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.

Recellularization of Bioengineered Scaffolds for Vascular Composite Allotransplantation.

Traumatic injuries or cancer resection resulting in large volumetric soft tissue loss requires surgical reconstruction. Vascular composite allotransplantation (VCA) is an emerging reconstructive option that transfers multiple, complex tissues as a whole subunit from donor to recipient. Although promising, VCA is limited due to side effects of immunosuppression. Tissue-engineered scaffolds obtained by decellularization and recellularization hold great promise. Decellularization is a process that removes cellular materials while preserving the extracellular matrix architecture. Subsequent recellularization of these acellular scaffolds with recipient-specific cells can help circumvent adverse immune-mediated host responses and allow transplantation of allografts by reducing and possibly eliminating the need for immunosuppression. Recellularization of acellular tissue scaffolds is a technique that was first investigated and reported in whole organs. More recently, work has been performed to apply this technique to VCA. Additional work is needed to address barriers associated with tissue recellularization such as: cell type selection, cell distribution, and functionalization of the vasculature and musculature. These factors ultimately contribute to achieving tissue integration and viability following allotransplantation. The present work will review the current state-of-the-art in soft tissue scaffolds with specific emphasis on recellularization techniques. We will discuss biological and engineering process considerations, technical and scientific challenges, and the potential clinical impact of this technology to advance the field of VCA and reconstructive surgery.

Two new applications in the study of angiogenesis the CAM assay: Acellular scaffolds and organoids.

The chick embryo chorioallantoic membrane (CAM) is a rich vascularized extraembryonic membrane that is commonly used as an in vivo experimental model to study molecules with angiogenic and anti-angiogenic activity, tumor growth and metastasis. Among other applications of the CAM assay, more recently this assay has been used for the study of acellular scaffolds and of organoids, and of their angiogenic capacity. The aim of this review article is to summarize the literature data concerning these two new applications of the CAM assay and to underline the advantages of this assay.

Therapeutic Acellular Scaffolds for Limiting Left Ventricular Remodelling-Current Status and Future Directions.

Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Following MI, the hypoxic microenvironment triggers apoptosis, disrupts the extracellular matrix and forms a non-functional scar that leads towards adverse left ventricular (LV) remodelling. If left untreated this eventually leads to heart failure. Besides extensive advancement in medical therapy, complete functional recovery is never accomplished, as the heart possesses limited regenerative ability. In recent decades, the focus has shifted towards tissue engineering and regenerative strategies that provide an attractive option to improve cardiac regeneration, limit adverse LV remodelling and restore function in an infarcted heart. Acellular scaffolds possess attractive features that have made them a promising therapeutic candidate. Their application in infarcted areas has been shown to improve LV remodelling and enhance functional recovery in post-MI hearts. This review will summarise the updates on acellular scaffolds developed and tested in pre-clinical and clinical scenarios in the past five years with a focus on their ability to overcome damage caused by MI. It will also describe how acellular scaffolds alone or in combination with biomolecules have been employed for MI treatment. A better understanding of acellular scaffolds potentialities may guide the development of customised and optimised therapeutic strategies for MI treatment.

Preclinical Testing of Living Tissue-Engineered Heart Valves for Pediatric Patients, Challenges and Opportunities.

Introduction: Pediatric patients with cardiac congenital diseases require heart valve implants that can grow with their natural somatic increase in size. Current artificial valves perform poorly in children and cannot grow; thus, living-tissue-engineered valves capable of sustaining matrix homeostasis could overcome the current drawbacks of artificial prostheses and minimize the need for repeat surgeries. Materials and Methods: To prepare living-tissue-engineered valves, we produced completely acellular ovine pulmonary valves by perfusion. We then collected autologous adipose tissue, isolated stem cells, and differentiated them into fibroblasts and separately into endothelial cells. We seeded the fibroblasts in the cusp interstitium and onto the root adventitia and the endothelial cells inside the lumen, conditioned the living valves in dedicated pulmonary heart valve bioreactors, and pursued orthotopic implantation of autologous cell-seeded valves with 6 months follow-up. Unseeded valves served as controls. Results: Perfusion decellularization yielded acellular pulmonary valves that were stable, no degradable in vivo, cell friendly and biocompatible, had excellent hemodynamics, were not immunogenic or inflammatory, non thrombogenic, did not calcify in juvenile sheep, and served as substrates for cell repopulation. Autologous adipose-derived stem cells were easy to isolate and differentiate into fibroblasts and endothelial-like cells. Cell-seeded valves exhibited preserved viability after progressive bioreactor conditioning and functioned well in vivo for 6 months. At explantation, the implants and anastomoses were intact, and the valve root was well integrated into host tissues; valve leaflets were unchanged in size, non fibrotic, supple, and functional. Numerous cells positive for a-smooth muscle cell actin were found mostly in the sinus, base, and the fibrosa of the leaflets, and most surfaces were covered by endothelial cells, indicating a strong potential for repopulation of the scaffold. Conclusions: Tissue-engineered living valves can be generated in vitro using the approach described here. The technology is not trivial and can provide numerous challenges and opportunities, which are discussed in detail in this paper. Overall, we concluded that cell seeding did not negatively affect tissue-engineered heart valve (TEHV) performance as they exhibited as good hemodynamic performance as acellular valves in this model. Further understanding of cell fate after implantation and the timeline of repopulation of acellular scaffolds will help us evaluate the translational potential of this technology.

Platforms to test the temporospatial capabilities of carrier systems in delivering growth factors to benefit vascular bioengineering.

In this study we produced a set of in vitro culture platforms to model vascular cell responses to growth factors and factor delivery vehicles. Two of the systems (whole vessel and whole lung vascular development) were supported by microfluidic systems facilitating media circulation and waste removal. We assessed vascular endothelial growth factor (VEGF) delivery by Pluronic F-127 hydrogel, 30 nm pore-sized microparticles (MPs), 60 nm pore-sized MP or a 50/50 mixture of 30 and 60 nm pore-sized MP. VEGF was delivered to porcine acellular lung vascular scaffolds (2.5 cm2 square pieces or whole 3D segments of acellular blood vessels) as well as whole acellular lung scaffolds. Scaffold-cell attachment was examined as was vascular tissue formation. We showed that a 50/50 mixture of 30 and 60 nm pore-sized silicon wafer MPs allowed for long-term release of VEGF within the scaffold vasculature and supported vascular endothelial tissue development during in vitro culture.

Self-assembling Peptide Hydrogels Facilitate Vascularization in Two-Component Scaffolds.

One of the major constraints against using polymeric scaffolds as tissue-regenerative matrices is a lack of adequate implant vascularization. Self-assembling peptide hydrogels can sequester small molecules and biological macromolecules, and they can support infiltrating cells in vivo. Here we demonstrate the ability of self-assembling peptide hydrogels to facilitate angiogenic sprouting into polymeric scaffolds after subcutaneous implantation. We constructed two-component scaffolds that incorporated microporous polymeric scaffolds and viscoelastic nanoporous peptide hydrogels. Nanofibrous hydrogels modified the biocompatibility and vascular integration of polymeric scaffolds with microscopic pores (pore diameters: 100-250 µm). In spite of similar amphiphilic sequences, charges, secondary structures, and supramolecular nanostructures, two soft hydrogels studied herein had different abilities to aid implant vascularization, but had similar levels of cellular infiltration. The functional difference of the peptide hydrogels was predicted by the difference in the bioactive moieties inserted into the primary sequences of the peptide monomers. Our study highlights the utility of soft supramolecular hydrogels to facilitate host-implant integration and control implant vascularization in biodegradable polyester scaffolds in vivo. Our study provides useful tools in designing multi-component regenerative scaffolds that recapitulate vascularized architectures of native tissues.

Angiogenic hydrogels for dental pulp revascularization.

Angiogenesis is critical for tissue healing and regeneration. Promoting angiogenesis in materials implanted within dental pulp after pulpectomy is a major clinical challenge in endodontics. We demonstrate the ability of acellular self-assembling peptide hydrogels to create extracellular matrix mimetic architectures that guide in vivo development of neovasculature and tissue deposition. The hydrogels possess facile injectability, as well as sequence-level functionalizability. We explore the therapeutic utility of an angiogenic hydrogel to regenerate vascularized pulp-like soft tissue in a large animal (canine) orthotopic model. The regenerated soft tissue recapitulates key features of native pulp, such as blood vessels, neural filaments, and an odontoblast-like layer next to dentinal tubules. Our study establishes angiogenic peptide hydrogels as potent scaffolds for promoting soft tissue regeneration in vivo. STATEMENT OF SIGNIFICANCE: A major challenge to endodontic tissue engineering is the lack of in situ angiogenesis within intracanal implants, especially after complete removal of the dental pulp. The lack of a robust vasculature in implants limit integration of matrices with the host tissue and regeneration of soft tissue. We demonstrate the development of an acellular material that promotes tissue revascularization in vivo without added growth factors, in a preclinical canine model of pulp-like soft-tissue regeneration. Such acellular biomaterials would facilitate pulp revascularization approaches in large animal models, and translation into human clinical trials.

Decellularization techniques and their applications for the repair and regeneration of the nervous system.

A variety of surgical and non-surgical approaches have been used to address the impacts of nervous system injuries, which can lead to either impairment or a complete loss of function for affected patients. The inherent ability of nervous tissues to repair and/or regenerate is dampened due to irreversible changes that occur within the tissue remodeling microenvironment following injury. Specifically, dysregulation of the extracellular matrix (i.e., scarring) has been suggested as one of the major factors that can directly impair normal cell function and could significantly alter the regenerative potential of these tissues. A number of tissue engineering and regenerative medicine-based approaches have been suggested to intervene in the process of remodeling which occurs following injury. Decellularization has become an increasingly popular technique used to obtain acellular scaffolds, and their derivatives (hydrogels, etc.), which retain tissue-specific components, including critical structural and functional proteins. These advantageous characteristics make this approach an intriguing option for creating materials capable of stimulating the sensitive repair mechanisms associated with nervous system injuries. Over the past decade, several diverse decellularization methods have been implemented specifically for nervous system applications in an attempt to carefully remove cellular content while preserving tissue morphology and composition. Each application-based decellularized ECM product requires carefully designed treatments that preserve the unique biochemical signatures associated within each tissue type to stimulate the repair of brain, spinal cord, and peripheral nerve tissues. Herein, we review the decellularization techniques that have been applied to create biomaterials with the potential to promote the repair and regeneration of tissues within the central and peripheral nervous system.

Preclinical studies of acellular extracellular matrices as small-caliber vascular grafts.

Due to morbidity and mortality of cardiovascular diseases around the globe, there has been an unmet clinical need for small caliber vascular grafts. Autologous vessels are still the gold standard for small caliber vascular grafts (<6 mm). In an attempt to develop a tissue-engineered vascular graft, several approaches have been pursued. One of the promising techniques is the use of acellular matrices offering a prospect of being able to meet the demand for small caliber vessels. Acellular matrices can ideally preserve the vascular wall complexity, biochemical properties, and bioactivity required for tissue regeneration and function. Various strategies have emerged to increase long term patency of acellular matrices including surface modification and pre-implantation cell seeding. This article reviews the most recent and relevant in vivo studies on acellular small caliber vascular grafts, which provides an outlook toward the preclinical potential of acellular extracellular matrices in vascular tissue engineering.

[Acellular vascular scaffolds modified by ammonium phosphate zwitterions can effectively resist thrombosis and promote endothelialization in vitro].

Due to limited availability of autologous blood vessels (blood vessels from the same recipient used for vascular transplantation materials) and inadequate growth ability of non-autologous blood vessels (artificial blood vessel transplantation materials), more and more attention has been paid to tissue engineering blood vessels. In this study, we constructed an ammonium phosphate zwitterion modified acellular vascular scaffold with highly biocompatible bone marrow-derived endothelial progenitor cells as the inner layer of a new vascular transplantation material. The vascular acellular scaffolds were modified by a simple method-co-precipitation method. The platelet adhesion test, hemolysis test, recalcification test and cytotoxicity of acellular vascular scaffolds in vitro were evaluated. Ammonium phosphate zwitterions modified endothelial progenitor cells on the surface of acellular scaffolds with concave and convex structure on the surface of natural vascular lumen can be effectively promoted by improving anticoagulant activity. Modified acellular scaffolds have similar mechanical properties to natural blood vessels and can effectively construct endothelialization in vitro. The results of this study provide a preliminary exploration for the modification of vascular acellular scaffolds to achieve anti-thrombosis and endothelialization in vitro.

Comparison of various reagents for preparing a decellularized porcine cartilage scaffold.

Cartilage lesion repair is difficult due to the limited self-repair capability of cartilage and its lack of vascularization. Our previous study established a sandwich model for engineering cartilage with acellular cartilage sheets (ACSs) and chondrocytes. However, there is still debate over which agent achieves the optimal decellularization of cartilage sheets. In addition, changes in the extracellular matrix after decellularization are worth studying. We aimed to determine the optimal decellularization reagents and decellularization time for preparing cartilage sheets. This study compared the effects of 2 extraction chemicals [t-octylphenoxypolyethoxyethanol (Triton X-100) and sodium dodecyl sulfate (SDS)] on cartilage sheets. The sheets were soaked in various concentrations (0.1-2%) of the extraction solutions for various time periods (24-72 h). After the decellularization process with the various treatments, we examined the cell removal and preservation of the matrix components and microstructure to determine which method was the most efficient while inducing minimal damage to the perichondrium. Both protocols achieved decellularization within an acceptable time. DNA analysis showed that the reagent removed nearly all of the DNA from the cartilage sheets. The growth factor contents in the Triton X-100 samples were higher than those in the SDS samples, quantified by enzyme-linked immunosorbent assay (ELISA). Furthermore, Triton X-100 decreased the glycosaminoglycan (GAG) and increased the chondromodulin-I contents compared with SDS. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that the ACSs were not cytotoxic. In conclusion, our results demonstrate that cartilage sheets decellularized by 1% SDS for 24 h or by 2% Triton X-100 for 48 h may be suitable candidate scaffolds for cartilage tissue engineering.

Acellular vascular scaffolds modified by ammonium phosphate zwitterions can effectively resist thrombosis and promote endothelialization in vitro / 生物工程学报

Due to limited availability of autologous blood vessels (blood vessels from the same recipient used for vascular transplantation materials) and inadequate growth ability of non-autologous blood vessels (artificial blood vessel transplantation materials), more and more attention has been paid to tissue engineering blood vessels. In this study, we constructed an ammonium phosphate zwitterion modified acellular vascular scaffold with highly biocompatible bone marrow-derived endothelial progenitor cells as the inner layer of a new vascular transplantation material. The vascular acellular scaffolds were modified by a simple method-co-precipitation method. The platelet adhesion test, hemolysis test, recalcification test and cytotoxicity of acellular vascular scaffolds in vitro were evaluated. Ammonium phosphate zwitterions modified endothelial progenitor cells on the surface of acellular scaffolds with concave and convex structure on the surface of natural vascular lumen can be effectively promoted by improving anticoagulant activity. Modified acellular scaffolds have similar mechanical properties to natural blood vessels and can effectively construct endothelialization in vitro. The results of this study provide a preliminary exploration for the modification of vascular acellular scaffolds to achieve anti-thrombosis and endothelialization in vitro.

Tissue Engineering Strategies for Myocardial Regeneration: Acellular Versus Cellular Scaffolds?

Heart disease remains one of the leading causes of death in industrialized nations with myocardial infarction (MI) contributing to at least one fifth of the reported deaths. The hypoxic environment eventually leads to cellular death and scar tissue formation. The scar tissue that forms is not mechanically functional and often leads to myocardial remodeling and eventual heart failure. Tissue engineering and regenerative medicine principles provide an alternative approach to restoring myocardial function by designing constructs that will restore the mechanical function of the heart. In this review, we will describe the cellular events that take place after an MI and describe current treatments. We will also describe how biomaterials, alone or in combination with a cellular component, have been used to engineer suitable myocardium replacement constructs and how new advanced culture systems will be required to achieve clinical success.

Porcine kidneys as a source of ECM scaffold for kidney regeneration.

OBJECTIVE: To produce and examine decellularized kidney scaffolds from porcine as a platform for kidney regeneration research. METHODS: Porcine kidneys were decellularized with sodium dodecyl sulfate solution and Triton X-100 after the blood was rinsed. Then the renal ECM scaffolds were examined for vascular imaging, histology to investigate the vascular patency, degree of decellularization. RESULTS: Renal ECM scaffolds of porcine kidneys were successfully produced. Decellularized renal scaffolds retained intact microarchitecture including the renal vasculature and essential extracellular matrix components. CONCLUSION: We have developed an excellent decellularization method that can be used in large organs. These scaffolds maintain their basic components, and show intact vasculature system. This represents a step toward development of a transplantable organ using tissue engineering techniques.

Platelet lysate embedded scaffolds for skin regeneration.

INTRODUCTION: The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems. METHODS: The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion. RESULTS: Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure. CONCLUSION: Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing.

Acellular spinal cord scaffold seeded with bone marrow stromal cells protects tissue and promotes functional recovery in spinal cord-injured rats.

Therapy using scaffolds seeded with stem cells plays an important role in repair of spinal cord injury (SCI), with the transplanted cells differentiating into nerve cells to replace the lost tissue while releasing neurotrophic factors that contribute to repair following SCI and enhance the function of the damaged nervous system. The present study investigated the ability to extend the survival time of bone marrow stromal cells (BMSCs) to restore the damaged spinal cord and improve functional recovery by grafting acellular spinal cord (ASC) scaffold seeded or not with BMSCs in a rat model of acute hemisected SCI. BBB scores revealed that treatment with BMSCs seeded into ASC scaffold led to an obvious improvement in motor function recovery compared with treatment with ASC scaffold alone or untreated controls. This improvement was evident at 2 and 8 weeks after surgery (P < 0.05). When BMSCs labeled with 5-bromodeoxyuridine were implanted together with ASC scaffold into the injured sites, they differentiated into glial cells, and some BMSCs could be observed within the graft by immunofluorescent staining at 8 weeks after implantation. Evaluation of caspase-3 activation suggested that the graft group was able to reduce apoptosis compared with SCI alone at 8 weeks after operation (P < 0.05). This study suggests that ASC scaffolds have the ability to enhance BMSC survival and improve differentiation and could also reduce native damaged nerve tissue apoptosis, thus protecting host tissue as well as improving functional recovery after implantation.