Supramolecular assembly of multi-purpose tissue engineering platforms from human extracellular matrix.

Simple SummaryHuman breast tissue extracellular matrix (ECM) is a microenvironment essential for the survival and biological activities of mammary epithelial cells. The ECM structural features of human breast tissues remain poorly defined. In this study, we identified the structural and mechanical properties of human normal breast and invasive ductal carcinoma tissue ECM using histological methods and atomic force microscopy. Additionally, a protein hydrogel was generated using human breast tissue ECM and defined for its microstructural features using immunofluorescence imaging and machine learning. Furthermore, we examined the three-dimensional growth of normal mammary epithelial cells or breast cancer cells cultured on the ECM protein hydrogel, where the cells exhibited biological phenotypes like those seen in native breast tissues. Our data provide novel insights into cancer cell biology, tissue microenvironment mimicry and engineering, and native tissue ECM-based biomedical and pharmaceutical applications.Tissue extracellular matrix (ECM) is a structurally and compositionally unique microenvironment within which native cells can perform their natural biological activities. Cells grown on artificial substrata differ biologically and phenotypically from those grown within their native tissue microenvironment. Studies examining human tissue ECM structures and the biology of human tissue cells in their corresponding tissue ECM are lacking. Such investigations will improve our understanding about human pathophysiological conditions for better clinical care. We report here human normal breast tissue and invasive ductal carcinoma tissue ECM structural features. For the first time, a hydrogel was successfully fabricated using whole protein extracts of human normal breast ECM. Using immunofluorescence staining of type I collagen (Col I) and machine learning of its fibrous patterns in the polymerized human breast ECM hydrogel, we have defined the microstructural characteristics of the hydrogel and compared the microstructures with those of other native ECM hydrogels. Importantly, the ECM hydrogel supported 3D growth and cell-ECM interaction of both normal and cancerous mammary epithelial cells. This work represents further advancement toward full reconstitution of the human breast tissue microenvironment, an accomplishment that will accelerate the use of human pathophysiological tissue-derived matrices for individualized biomedical research and therapeutic development.

Osteochondral injuries are challenging to repair due to their complex tissue anatomy and restricted self-repairing ability associated with a limited blood supply. Osteochondral tissue engineering is an important clinical aspect of the management and treatment of cartilage and underlying bone. In the present study, we fabricated human placenta-derived extracellular matrix sponges (PEMS) for repair of osteochondral tissue through a decellularization process. There were no significant cellular components present in the PEMS; hematoxylin & eosin/DAPI staining, DNA quantification and agarose gel electrophoresis were used to evaluate the extent of decellularization. Moreover, no significant alteration to the collagen and glycosaminoglycan (native extracellular matrix) content of the PEMS was observed. PEMS in vitro provided a non-cytotoxic environment rich in bioactive cues for human amniotic membrane-derived stem cells (HAMSCs) to proliferate in and differentiate into chondrogenic and osteogenic lineages under induction. Histological analysis at 28 days after the PEMS were subcutaneously implanted demonstrated no severe immune response in the host and supported the formation of blood vessels. To assess the osteochondral tissue repair ability of PEMS, cell-free PEMS (CFP) and cell-seeded PEMS (CSP) were implanted at osteochondral defect sites in a rabbit model. Histological scores indicated that osteochondral regeneration was more successful in the defects filled with CSP compared to those filled with CFP and empty defects (ED) after 60 days of implantation. In summary, a naturally derived biocompatible scaffold composed of extracellular matrix from human placenta has been successfully developed for osteochondral tissue engineering.

Robot-assisted orthotopic and heterotopic ovarian tissue transplantation techniques: surgical advances since our first success in 2000

Engineering Biomimetic Culture Systems: Impact On Human Bone Marrow-Derived Stem Cells.

Biological scaffolds composed of naturally occurring extracellular matrix (ECM) have been utilized as templates for the constructive remodelling of numerous tissues in preclinical studies and human clinical applications. The mechanisms by which ECM induces constructive remodelling are not well understood, but it appears that the degradation products of ECM scaffolds may play key roles in cell recruitment. The objective of the present study was to investigate the effects of age and species of the tissue from which ECM is harvested on the chemoattractant activity of degradation products of ECM for human keratinocyte stem and progenitor cells. Adult human skin ECM, fetal human skin ECM and adult porcine skin ECM were prepared, enzymatically digested, characterized by SDS-PAGE and evaluated for in vitro chemoattractant activity for human keratinocyte progenitor and stem cells (HEKn). Degradation products of human fetal skin ECM showed greater chemoattractant activity than human adult skin ECM degradation products for the HEKn. Degradation products of porcine adult skin ECM showed greater chemoattractant activity than human adult skin ECM. The human fetal skin ECM degradation products showed the strongest chemoattractant activity for the HEKn. The findings of this study support the concept that the mechanism of ECM scaffold remodelling involves the recruitment of lineage-directed progenitor cells by scaffold degradation products, and that both the age and species of the tissue from which the ECM is harvested have an effect upon this chemoattractant potential.

Machine learning identifies remodeling patterns in human lung extracellular matrix.

Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however, inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study, we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization, the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance, only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel, contained no vitronectin but did contain collagen XII, ig-h3 and novel for hESC-supporting human matrices, substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however, distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus, human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings.

A great variety of natural and synthetic polymer materials have been utilized in soft tissue engineering as extracellular matrix (ECM) materials. Natural polymers, such as collagen and fibrin hydrogels, have experienced especially broad adoption due to the high density of cell adhesion sites compared to their synthetic counterparts, ready availability, and ease of use. However, these and other hydrogels lack the structural and mechanical anisotropy that define the ECM in many tissues, such as skeletal and cardiac muscle, tendon, and cartilage. Herein, we present a facile, low-cost, and automated method of preparing collagen microfibers, organizing these fibers into precisely controlled mesh designs, and embedding these meshes in a bulk hydrogel, creating a composite biomaterial suitable for a wide variety of tissue engineering and regenerative medicine applications. With the assistance of custom software tools described herein, mesh patterns are designed by a digital graphical user interface and translated into protocols that are executed by a custom mesh collection and organization device. We demonstrate a high degree of precision and reproducibility in both fiber and mesh fabrication, evaluate single fiber mechanical properties, and provide evidence of collagen self-assembly in the microfibers under standard cell culture conditions. This work offers a powerful, flexible platform for the study of tissue engineering and cell material interactions, as well as the development of therapeutic biomaterials in the form of custom collagen microfiber patterns that will be accessible to all through the methods and techniques described here.Impact StatementCollagen microfiber meshes have immediate and broad applications in tissue engineering research and show high potential for later use in clinical therapeutics due to their compositional similarities to native extracellular matrix and tunable structural and mechanical characteristics. Physical and biological characterizations of these meshes demonstrate physiologically relevant mechanical properties, native-like collagen structure, and cytocompatibility. The methods presented herein not only describe a process through which custom collagen microfiber meshes can be fabricated but also provide the reader with detailed device plans and software tools to produce their own bespoke meshes through a precise, consistent, and automated process.

To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model. Ten chinchilla bastard rabbit corneas were exposed to a circular half-thickness keratotomy with a 3.0 mm diameter at the central cornea. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 K IU/ml) and 0.3% sodium dodecyl sulphate (SDS). The 3.0 mm in diameter decellularized corneal stromal xenograft was inserted into the pocket, and the incision was closed with four 10.0 nylon sutures. Clinical photographs were taken at day 1, day 7, day 30 and on a monthly basis for up to 6 months after transplantation. Six months after surgery, the rabbits were killed and eyes were enucleated. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the complete removal of the corneal cells after decellularization of porcine corneas and repopulation with rabbit cells. Alcian blue staining was performed for analysing the structure of the extracellular matrix (ECM). Efficient elimination of porcine cells was achieved by our decellularization protocol and confirmed via HE and DAPI stainings. Moreover, the major histoarchitectural ECM structure had been maintained as visualized by the alcian blue stain. Finally, the PSM was biocompatible with the host's epithelium evidenced as a regrowth covering the exposed xenograft. This novel technique of tissue engineering may provide one of many solutions to addressing anterior corneal pathological conditions in the face of a shortage of human corneal material.

Figure S1. Attachment and growth of clonal-density primary human melanocytes on ECM. Figure S2. Presence and requirement for laminin-332. Figure S3. Clonal survival of UV-irradiated primary human melanocytes after UVB exposure. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Tissue engineering, which is a promising approach for treating mankind, aims to repair, replace, maintain, or improve the function of damaged tissue or organ. Electrospun nanofibers, providing a three- dimensional environment to mimic the natural extracellular matrix (ECM), play an essential role in tissue engineer- ing as well as regenerative medicine. In this study we demonstrated the importance of scaffold fabrication method together with the effect of nanotopogeraphy to support the growth of cells. Two scaffolds were made from the bio- degradable and biocompatible poly (lactic-co-glycolic acid) (PLGA) polymer by electrospinning and freeze-drying techniques respectively and the effectiveness in providing support for cell behavior was investigated. The scaffold structures were compared by scanning electron microscope (SEM). Fibroblast cell culture test was carried to investi- gate cell behaviors by inverted microscopic images. To investigate the effect of scaffolds on cell proliferation, we performed cell attachment experiments: 4',6-diamidino-2-phenylindole (DAPI) staining and 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyltetrazolium bromide (MTT). Our results showed that electrospun fibrous PLGA scaffolds provided more suitable surface for cell functioning compared with freeze-dried porous PLGA scaffolds, due to the feature similarity of the former to the native ECM. © Global Scientific Publishers 2013

In vascular tissue engineering strategies, the addition of vascular-specific extracellular matrix (ECM) components may better mimic the in vivo microenvironment and potentially enhance cell–matrix interactions and subsequent tissue growth. For this purpose, the exact composition of the human vascular ECM first needs to be fully characterized. Most research has focused on characterizing ECM components in mature vascular tissue; however, the developing fetal ECM matches the active environment required in vascular tissue engineering more closely. Consequently, we characterized the ECM protein composition of active (fetal) and quiescent (mature) renal arteries using a proteome analysis of decellularized tissue. The obtained human fetal renal artery ECM proteome dataset contains higher levels of 15 ECM proteins versus the mature renal artery ECM proteome, whereas 16 ECM proteins showed higher levels in the mature tissue compared to fetal. Elastic ECM proteins EMILIN1 and FBN1 are significantly enriched in fetal renal arteries and are mainly produced by cells of mesenchymal origin. We functionally tested the role of EMILIN1 and FBN1 by anchoring the ECM secreted by vascular smooth muscle cells (SMCs) to glass coverslips. This ECM layer was depleted from either EMILIN1 or FBN1 by using siRNA targeting of the SMCs. Cultured endothelial cells (ECs) on this modified ECM layer showed alterations on the transcriptome level of multiple pathways, especially the Rho GTPase controlled pathways. However, no significant alterations in adhesion, migration or proliferation were observed when ECs were cultured on EMILIN1- or FNB1-deficient ECM. To conclude, the proteome analysis identified unique ECM proteins involved in the embryonic development of renal arteries. Alterations in transcriptome levels of ECs cultured on EMILIN1- or FBN1-deficient ECM showed that these candidate proteins could affect the endothelial (regenerative) response.

As the cornerstone of tissue engineering and regeneration medicine research, developing a cost-effective and bionic extracellular matrix (ECM) that can precisely modulate cellular behavior and form functional tissue remains challenging. An artificial ECM combining polysaccharides and fibrillar proteins to mimic the structure and composition of natural ECM provides a promising solution for cardiac tissue regeneration. In this study, we developed a bionic hydrogel scaffold by combining a quaternized β-chitin derivative (QC) and fibrin-matrigel (FM) in different ratios to mimic a natural ECM. We evaluated the stiffness of those composite hydrogels with different mixing ratios and their effects on the growth of human umbilical vein endothelial cells (HUVECs). The optimal hydrogels, QCFM1 hydrogels were further applied to load HUVECs into nude mice for in vivo angiogenesis. Besides, we encapsulated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) into QCFM hydrogels and employed 3D bioprinting to achieve batch fabrication of human-engineered heart tissue (hEHT). Finally, the myocardial structure and electrophysiological function of hEHT were evaluated by immunofluorescence and optical mapping. Designed artificial ECM has a tunable modulus (220-1380 Pa), which determines the different cellular behavior of HUVECs when encapsulated in these. QCFM1 composite hydrogels with optimal stiffness (800 Pa) and porous architecture were finally identified, which could adapt for in vitro cell spreading and in vivo angiogenesis of HUVECs. Moreover, QCFM1 hydrogels were applied in 3D bioprinting successfully to achieve batch fabrication of both ring-shaped and patch-shaped hEHT. These QCFM1 hydrogels-based hEHTs possess organized sarcomeres and advanced function characteristics comparable to reported hEHTs. The chitin-derived hydrogels are first used for cardiac tissue engineering and achieve the batch fabrication of functionalized artificial myocardium. Specifically, these novel QCFM1 hydrogels provided a reliable and economical choice serving as ideal ECM for application in tissue engineering and regeneration medicine.

Chapter 2 - Tissue repair with natural extracellular matrix (ECM) scaffolds

The circumcision of males is emphatically linked to numerous sexual dysfunctions. Many of the purported benefits do not hold up to the scrutiny of extensive literature surveys. Involuntary circumcision, particularly when not medically warranted, is also associated with many psychological and emotional traumas. Current methods to reconstruct the ablated tissue have significant drawbacks and produce a simple substitute that merely imitates the natural foreskin. Extracellular matrix–based scaffolds have been shown to be highly effective in the repair and regeneration of soft tissues; however, due to the unique nature of the foreskin tissue, commercially available biomaterial scaffolds would yield poor results. Therefore, this study discusses the development and evaluation of a tissue engineering scaffold derived from decellularized human foreskin extracellular matrix for foreskin reconstruction. A chemicophysical decellularization method was applied to human foreskin samples, sourced from consenting adult donors. The resulting foreskin dermal matrices were analyzed for their suitability for tissue engineering purposes, by biological, histological, and mechanical assessment; fresh frozen foreskin was used as a negative control. Sterility of samples at all stages was ensured by microbiological analysis. MTT assay was used to evaluate the absence of viable cells, and histological analysis was used to confirm the maintenance of the extracellular matrix structure and presence/integrity of collagen fibers. Bioactivity was determined by submitting tissue extracts to enzyme-linked immunosorbent assay and quantifying basic fibroblast growth factor content. Mechanical properties of the samples were determined using tensile stress tests. Results found foreskin dermal matrices were devoid of viable cells (p < 0.0001) and the matrix of foreskin dermal matrices was maintained. Basic fibroblast growth factor content doubled within after decellularization (p < 0.0001). Tensile stress tests found no statistically significant differences in the mechanical properties (p < 0.05). These results indicate that the derived foreskin dermal matrix may be suitable in a regenerative approach in the reconstruction of the human foreskin.