Acellular Human Placenta Small-Diameter Vessels as a Favorable Source of Super-Microsurgical Vascular Replacements: A Proof of Concept.

In this study, we aimed to evaluate the human placenta as a source of blood vessels that can be harvested for vascular graft fabrication in the submillimeter range. Our approach included graft modification to prevent thrombotic events. Submillimeter arterial grafts harvested from the human placenta were decellularized and chemically crosslinked to heparin. Graft performance was evaluated using a microsurgical arteriovenous loop (AVL) model in Lewis rats. Specimens were evaluated through hematoxylin-eosin and CD31 staining of histological sections to analyze host cell immigration and vascular remodeling. Graft patency was determined 3 weeks after implantation using a vascular patency test, histology, and micro-computed tomography. A total of 14 human placenta submillimeter vessel grafts were successfully decellularized and implanted into AVLs in rats. An appropriate inner diameter to graft length ratio of 0.81 ± 0.16 mm to 7.72 ± 3.20 mm was achieved in all animals. Grafts were left in situ for a mean of 24 ± 4 days. Decellularized human placental grafts had an overall patency rate of 71% and elicited no apparent immunological responses. Histological staining revealed host cell immigration into the graft and re-endothelialization of the vessel luminal surface. This study demonstrates that decellularized vascular grafts from the human placenta have the potential to serve as super-microsurgical vascular replacements.

Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering.

BACKGROUND: There is a great clinical need and it remains a challenge to develop artificial soft tissue constructs that can mimic the biomechanical properties and bioactivity of natural tissue. This is partly due to the lack of suitable biomaterials. Hydrogels made from human placenta offer high bioactivity and represent a potential solution to create animal-free 3D bioprinting systems that are both sustainable and acceptable, as placenta is widely considered medical waste. A combination with silk and gelatin polymers can bridge the biomechanical limitations of human placenta chorion extracellular matrix hydrogels (hpcECM) while maintaining their excellent bioactivity. METHOD: In this study, silk fibroin (SF) and tyramine-substituted gelatin (G-TA) were enzymatically crosslinked with human placental extracellular matrix (hpcECM) to produce silk-gelatin-ECM composite hydrogels (SGE) with tunable mechanical properties, preserved elasticity, and bioactive functions. The SGE composite hydrogels were characterized in terms of gelation kinetics, protein folding, and bioactivity. The cyto- and biocompatibility of the SGE composite was determined by in vitro cell culture and subcutaneous implantation in a rat model, respectively. The most cell-supportive SGE formulation was then used for 3-dimensional (3D) bioprinting that induced chemical crosslinking during extrusion. CONCLUSION: Addition of G-TA improved the mechanical properties of the SGE composite hydrogels and inhibited crystallization and subsequent stiffening of SF for up to one month. SGE hydrogels exhibit improved and tunable biomechanical properties and high bioactivity for encapsulated cells. In addition, its use as a bioink for 3D bioprinting with free reversible embedding of suspended hydrogels (FRESH) has been validated, opening the possibility to fabricate highly complex scaffolds for artificial soft tissue constructs with natural biomechanics in future.

Harnessing Human Decellularized Blood Vessel Matrices and Cellular Construct Implants to Promote Bone Healing in an Ex Vivo Organotypic Bone Defect Model.

Decellularized matrices offer a beneficial substitute for biomimetic scaffolds in tissue engineering. The current study examines the potential of decellularized placental vessel sleeves (PVS) as a periosteal protective sleeve to enhance bone regeneration in embryonic day 18 chick femurs contained within the PVS and cultured organotypically over a 10 day period. The femurs are inserted into decellularized biocompatibility-tested PVS and maintained in an organotypic culture for a period of 10 days. In femurs containing decellularized PVS, a significant increase in bone volume (p < 0.001) is evident, demonstrated by microcomputed tomography (µCT) compared to femurs without PVS. Histological and immunohistological analyses reveal extensive integration of decellularized PVS with the bone periosteum, and enhanced conservation of bone architecture within the PVS. In addition, the expressions of hypoxia inducible factor-1 alpha (HIF-1α), type II collagen (COL-II), and proteoglycans are observed, indicating a possible repair mechanism via a cartilaginous stage of the bone tissue within the sleeve. The use of decellularized matrices like PVS offers a promising therapeutic strategy in surgical tissue replacement, promoting biocompatibility and architecture of the tissue as well as a factor-rich niche environment with negligible immunogenicity.

An Effective Method of Atelocollagen Type 1/3 Isolation from Human Placenta and Its In Vitro Characterization in Two-Dimesional and Three-Dimensional Cell Culture Applications.

Pepsin-solubilized atelocollagen can be used to form highly complex three-dimensional matrices for a broad spectrum of tissue engineering applications. Moreover, it has a long history as a favorable biomaterial in pharmaceutical and medical industries. So far, the main sources for these approaches are collagens from xenogenic sources. Yet, these nonhuman collagens carry a risk of provoking immune reactions in patients. Here we describe an effective method of isolating atelocollagen type 1/3 (COL1/3) from human placenta. By combining a single pepsin digestion step with tangential flow filtration and further precipitation steps, we could purify COL1/3 within only 4 days of processing. The resulting COL1/3 was biochemically characterized by determining residual DNA content, proving the absence of impurities by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis combined with total amino acid quantification, identifying the isolated collagen types by Western blot analysis, and analyzing the spontaneous formation of fibrous structures on freeze-drying via scanning electron microscopy. Finally, the cytocompatibility of the isolated collagen was demonstrated in two dimensional using primary rat hepatocytes and in three dimensional by a sprouting assay of human umbilical vein endothelial cell. The isolation method described not only fulfills demands for cost-efficient bioengineering using a human waste material but also potentially increases overall safety for patients by use of homologous products.

Decellularized human placenta chorion matrix as a favorable source of small-diameter vascular grafts.

Biomaterials based on decellularized tissues are increasingly attracting attention as functional alternatives to other natural or synthetic materials. However, a source of non-cadaver human allograft material would be favorable. Here we establish a decellularization method of vascular tissue from cryopreserved human placenta chorionic plate starting with an initial freeze-thaw step followed by a series of chemical treatments applied with a custom-made perfusion system. This novel pulsatile perfusion set-up enabled us to successfully decellularize the vascular tissue with lower concentrations of chemicals and shorter exposure times compared to a non-perfusion process. The decellularization procedure described here lead to the preservation of the native extracellular matrix architecture and the removal of cells. Quantitative analysis revealed no significant changes in collagen content and a retained glycosaminoglycan content of approximately 29%. In strain-to-failure tests, the decellularized grafts showed similar mechanical behavior compared to native controls. In addition, the mechanical values for ultimate tensile strength and stiffness were in an acceptable range for in vivo applications. Furthermore, biocompatibility of the decellularized tissue and its recellularizationability to serve as an adequate substratum for upcoming recellularization strategies using primary human umbilical vein endothelial cells (HUVECs) was demonstrated. HUVECs cultured on the decellularized placenta vessel matrix performed endothelialization and maintained phenotypical characteristics and cell specific expression patterns. Overall, the decellularized human placenta vessels can be a versatile tool for experimental studies on vascularization and as potent graft material for future in vivo applications. STATEMENT OF SIGNIFICANCE: In the US alone more than 1million vascular grafts are needed in clinical practice every year. Despite severe disadvantages, such as donor site morbidity, autologous grafting from the patient's own arteries or veins is regarded as the gold standard for vascular tissue repair. Besides, strategies based on synthetic or natural materials have shown limited success. Tissue engineering approaches based on decellularized tissues are regarded as a promising alternative to clinically used treatments to overcome the observed limitations. However, a source for supply of non-cadaver human allograft material would be favorable. Here, we established a decellularization method of vascular tissue from the human placenta chorionic plate, a suitable human tissue source of consistent quality. The decellularized human placenta vessels can be a potent graft material for future in vivo applications and furthermore might be a versatile tool for experimental studies on vascularization.