Alignment of Cells and Extracellular Matrix Within Tissue- Engineered Substitutes

Most of the cells in our body are in direct contact with extracellular matrix (ECM) compo‐ nents which constitute a complex network of nano-scale proteins and glycosaminoglycans. Those cells constantly remodel the ECM by different processes. They build it by secreting dif‐ ferent proteins such as collagen, proteoglycans, laminins or degrade it by producing factors such as matrix metalloproteinase (MMP). Cells interact with the ECM via specific receptors, the integrins [1]. They also organize this matrix, guided by different stimuli, to generate pat‐ terns, essential for tissue and organ functions. Reciprocally, cells are guided by the ECM, they modify their morphology and phenotype depending on the protein types and organization via bidirectional integrin signaling [2-4]. In the growing field of tissue engineering [5], control of these aspects are of the utmost importance to create constructs that closely mimic native tis‐ sues. To do so, we must take into account the composition of the scaffold (synthetic, natural, biodegradable or not), its organization and the dimension of the structure. The particular alignment patterns of ECM and cells observed in tissues and organs such as the corneal stroma, vascular smooth muscle cells (SMCs), tendons, bones and skeletal mus‐ cles are crucial for organ function. SMCs express contraction proteins such as alpha-smoothmuscle (SM)-actin, desmin and myosin [6] that are essential for cell contraction [6]. To result in vessel contraction, the cells and ECM need to be organized in such a way that most cells are elongated in the same axis. For tubular vascular constructs, it is suitable that SMCs align in the circumferential direction, as they do in vivo [7, 8]. Another striking example of align‐ ment is skeletal muscle cells that form long polynuclear cells, all elongated in the same axis. Each cell generates a weak and short contraction pulse but collectively, it results in a strong, long and sustained contraction of the muscle and, in term, a displacement of the member. In the corneal stroma, the particular arrangement of the corneal fibroblasts (keratocytes) and ECM is essential to keep the transparency of this tissue [9-13]. Tendons also present a pecu‐ liar matrix alignment relative to the muscle axis. It gives a substantial resistance and excep‐ tional mechanical properties to the tissue in that axis [14, 15]. Intervertebral discs [16], cartilage [17], dental enamel [18], and basement membrane of epithelium are other examples of tissues/organs that present peculiar cell and matrix organization. By reproducing and controlling those alignment patterns within tissue-engineered substitutes, a more physiolog‐ ical representation of human tissues could be achieved. Taking into account the importance of cell microenvironment on the functionality of tissue engineered organ substitutes, one can assume the importance of being able to customise the 3D structure of the biomaterial or scaffold supporting cell growth. To do so, some methods have been developed and most of them rely on topographic or contact guidance. This is the phenomenon by which cells elongate and migrate in the same axis as the ECM. Topographic guidance was so termed by Curtis and Clark [19] to include cell shape, orientation and movement in the concept of contact guidance described by Harrison [20] and implemented by Weiss [21, 22]. Therefore, if one can achieve ECM alignment, cells will follow the same pattern. Inversely, if cells are aligned on a patterned culture plate, the end result would be aligned ECM deposition [23]. The specific property of tissues or materials that present a variation in their mechanical and structural properties in different axis is called anisotropy. This property can be evaluated ei‐ ther by birefringence measurements [24, 25], mechanical testing in different axis [26], immu‐ nological staining of collagen or actin filaments [23] or direct visualisation of collagen fibrils using their self-fluorescence around 488 nm [27, 28]. Several techniques have been recently developed to mimic the specific alignment of cells within tissues to produce more physiologically relevant constructs. In this chapter, we will describe five different techniques, collagen gel compaction, electromagnetic field, electro‐ spinning of nanofibers, mechanical stimulation and microstructured culture plates

Progress in developing a living human tissue-engineered tri-leaflet heart valve assembled from tissue produced by the self-assembly approach

The aortic heart valve is constantly subjected to pulsatile flow and pressure gradients which, associated with cardiovascular risk factors and abnormal hemodynamics (i.e. altered wall shear stress), can cause stenosis and calcification of the leaflets and result in valve malfunction and impaired circulation. Avail- able options for valve replacement include homograft, allogenic or xenogenic graft as well as the implan- tation of a mechanical valve. A tissue-engineered heart valve containing living autologous cells would represent an alternative option, particularly for pediatric patients, but still needs to be developed. The present study was designed to demonstrate the feasibility of using a living tissue sheet produced by the self-assembly method, to replace the bovine pericardium currently used for the reconstruction of a stented human heart valve. In this study, human fibroblasts were cultured in the presence of sodium ascorbate to produce tissue sheets. These sheets were superimposed to create a thick construct. Tissue pieces were cut from these constructs and assembled together on a stent, based on techniques used for commercially available replacement valves. Histology and transmission electron microscopy analysis showed that the fibroblasts were embedded in a dense extracellular matrix produced in vitro. The mechanical properties measured were consistent with the fact that the engineered tissue was resistant and could be cut, sutured and assembled on a wire frame typically used in bioprosthetic valve assembly. After a culture period in vitro, the construct was cohesive and did not disrupt or disassemble. The tissue engineered heart valve was stimulated in a pulsatile flow bioreactor and was able to sustain multiple duty cycles. This prototype of a tissue-engineered heart valve containing cells embedded in their own extracellular matrix and sewn on a wire frame has the potential to be strong enough to support physio- logical stress. The next step will be to test this valve extensively in a bioreactor and at a later date, in a large animal model in order to assess in vivo patency of the graft

In vivo remodeling of fibroblast-derived vascular scaffolds implanted for 6 months in rats

There is a clinical need for tissue-engineered small-diameter (<6 mm) vascular grafts since clinical applications are halted by the limited suitability of autologous or synthetic grafts. This study uses the self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold (FDVS) that can be available off-the-shelf. Briefly, extracellular matrix scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and decellularized by immersion in deionized water. The FDVSs were implanted as an aortic interpositional graft in six Sprague-Dawley rats for 6 months. Five out of the six implants were still patent 6 months after the surgery. Histological analysis showed the infiltration of cells on both abluminal and luminal sides, and immunofluorescence analysis suggested the formation of neomedia comprised of smooth muscle cells and lined underneath with an endothelium. Furthermore, to verify the feasibility of producing tissue-engineered blood vessels of clinically relevant length and diameter, scaffolds with a 4.6 mm inner diameter and 17 cm in length were fabricated with success and stored for an extended period of time, while maintaining suitable properties following the storage period. This novel demonstration of the potential of the FDVS could accelerate the clinical availability of tissue-engineered blood vessels and warrants further preclinical studies

Optimization of culture conditions for porcine corneal endothelial cells

Purpose : To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology. Methods : PCEC cultures were seeded at an initial cell density of 400 cells/cm2 in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm2 in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm2 in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 μg/ml), ascorbic acid (10, 20, 40 μg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology. Results : Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium. Conclusions : Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells

The Triplex BioValsalva Prostheses To Reconstruct the Aortic Valve and the Aortic Root

The Bentall procedure introduced in 1968 represents an undisputed cure to treat multiple pathologies involving the aortic valve and the ascending thoracic aorta. Over the years, multiple modifications have been introduced as well as a standardized approach to the operation with the goal to prevent long-term adverse events. The BioValsalva prosthesis provides a novel manner to more efficiently reconstruct the aortic valve together with the anatomy of the aortic root with the implantation of a valved conduit. This prosthesis comprises three sections: the collar supporting the valve; the skirt mimicking the Valsalva, which is suitable for the anastomoses with the coronary arteries; and the main body of the graft, which is designed to replace the ascending aorta. The BioValsalva prosthesis allows the Bentall operation to be used in patients whose aortic valve cannot be spared

The cumate gene-switch: a system for regulated expression in mammalian cells

BACKGROUND: A number of expression systems have been developed where transgene expression can be regulated. They all have specific characteristics making them more suitable for certain applications than for others. Since some applications require the regulation of several genes, there is a need for a variety of independent yet compatible systems. RESULTS: We have used the regulatory mechanisms of bacterial operons (cmt and cym) to regulate gene expression in mammalian cells using three different strategies. In the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a strong constitutive promoter. Addition of cumate, a small molecule, relieves the repression. In the transactivator configuration, a chimaeric transactivator (cTA) protein, formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to multiple copies of CuO, placed upstream of the CMV minimal promoter. Cumate addition abrogates DNA binding and therefore transactivation by cTA. Finally, an adenoviral library of cTA mutants was screened to identify a reverse cumate activator (rcTA), which activates transcription in the presence rather than the absence of cumate. CONCLUSION: We report the generation of a new versatile inducible expression system

Interleukin-10 controls the protective effects of circulating microparticles from patients with septic shock on tissue-engineered vascular media

During sepsis, inflammation can be orchestrated by the interaction between circulating and vascular cells that, under activation, release MPs (microparticles). Previously, we reported that increased circulating MPs in patients with sepsis play a pivotal role in ex vivo vascular function suggesting that they are protective against vascular hyporeactivity. The present study was designed to investigate the effects of MPs from patients with sepsis on the contractile response of TEVM (tissue-engineered vascular media). TEVM that were composed only of a media layer were produced by tissue engineering from human arterial SMCs (smooth muscle cells) isolated from umbilical cords. TEVM was incubated with MPs isolated from whole blood of 16 patients with sepsis. TEVM were incubated for 24 h with MPs and used for the study of vascular contraction, direct measurements of NO and O2- (superoxide anion) production by EPR and quantification of mRNA cytokine expression. MPs from patients with sepsis increased contraction induced by histamine in TEVM. This effect was not associated with inflammation, neither linked to the activation of NF-kappaB (nuclear factor kappaB) pathway nor to the increase in iNOS (inducible NO synthase) and COX (cyclo-oxygenase)-2 expression. In contrast, mRNA expression of IL (interleukin)-10 was enhanced. Then, we investigated the effect of IL-10 on vascular hyporeactivity induced by LPS (lipopolysaccharide). Although IL-10 treatment did not modify the contractile response in TEVM by itself, this interleukin restored contraction in LPS-treated TEVM. In addition, IL-10 treatment both prevented vascular hyporeactivity induced by LPS injection in mice and improved survival of LPS-injected mice. These findings show an association between the capacity of MPs from patients with sepsis to restore vascular hyporeactivity induced by LPS and their ability to increase IL-10 in the tissue-engineered blood vessel model

A Floating Thrombus Anchored at the Proximal Anastomosis of a Woven Thoracic Graft Mimicking a Genuine Aortic Dissection

An aortoesophageal fistula following surgery for a ruptured 6.6-cm thoracic aneurysm in a 69-yearold female was repaired using a 34-mm woven prosthetic graft. A follow-up computed tomography (CT) scan at 10 days postoperatively revealed a dissection-like picture in the region of the graft, which was treated conservatively. The patient eventually died from sepsis and multiorgan failure. At autopsy, the graft was retrieved in situ and studied by detailed gross, microscopy, and scanning electron microscopy (SEM) examination. Gross observation confirmed that the dissection resulted from the rolling of the internal capsule downstream. A massive thrombus anchored at the proximal anastomosis and held by a narrow head was also noted. The thrombus demonstrated reorganization in the area of the anastomosis, with a false lumen in its distal half. The reminder of the thrombus consisted of layered fibrin. After gross examination, the fabric graft was found to be flawless. Additional detailed studies were also done using microscopy, SEM, and gross examination

Analysis of baculovirus aggregates using flow cytometry

NRC publication: Ye

Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis

NRC publication: Ye