Regenerative medicine for oesophageal reconstruction after cancer treatment.

Removal of malignant tissue in patients with oesophageal cancer and replacement with autologous grafts from the stomach and colon can lead to problems. The need to reduce stenosis and anastomotic leakage after oesophagectomy is a high priority. Developments in tissue-engineering methods and cell-sheet technology have improved scaffold materials for oesophageal repair. Despite the many successful animal studies, few tissue-engineering approaches have progressed to clinical trials. In this Review, we discuss the status of oesophagus reconstruction after surgery. In particular, we highlight two clinical trials that used decellularised constructs and epithelial cell sheets to replace excised tissues after endoscopic submucosal dissection or mucosal resection procedures. Results from the trials showed that both decellularised grafts and epithelial-cell sheets prevented stenosis. By contrast, animal studies have shown that the use of tissue-engineered constructs after oesophagectomy remains a challenge.

Oligo[poly(ethylene glycol)fumarate] hydrogel enhances osteochondral repair in porcine femoral condyle defects.

BACKGROUND: Management of osteochondritis dissecans remains a challenge. Use of oligo[poly(ethylene glycol)fumarate] (OPF) hydrogel scaffold alone has been reported in osteochondral defect repair in small animal models. However, preclinical evaluation of usage of this scaffold alone as a treatment strategy is limited. QUESTIONS/PURPOSES: We therefore (1) determined in vitro pore size and mechanical stiffness of freeze-dried and rehydrated freeze-dried OPF hydrogels, respectively; (2) assessed in vivo gross defect filling percentage and histologic findings in defects implanted with rehydrated freeze-dried hydrogels for 2 and 4 months in a porcine model; (3) analyzed highly magnified histologic sections for different types of cartilage repair tissues, subchondral bone, and scaffold; and (4) assessed neotissue filling percentage, cartilage phenotype, and Wakitani scores. METHODS: We measured pore size of freeze-dried OPF hydrogel scaffolds and mechanical stiffness of fresh and rehydrated forms. Twenty-four osteochondral defects from 12 eight-month-old micropigs were equally divided into scaffold and control (no scaffold) groups. Gross and histologic examination, one-way ANOVA, and one-way Mann-Whitney U test were performed at 2 and 4 months postoperatively. RESULTS: Pore sizes ranged from 20 to 433 µm in diameter. Rehydrated freeze-dried scaffolds had mechanical stiffness of 1 MPa. The scaffold itself increased percentage of neotissue filling at both 2 and 4 months to 58% and 54%, respectively, with hyaline cartilage making up 39% of neotissue at 4 months. CONCLUSIONS: Rehydrated freeze-dried OPF hydrogel can enhance formation of hyaline-fibrocartilaginous mixed repair tissue of osteochondral defects in a porcine model. CLINICAL RELEVANCE: Rehydrated freeze-dried OPF hydrogel alone implanted into cartilage defects is insufficient to generate a homogeneously hyaline cartilage repair tissue, but its spacer effect can be enhanced by other tissue-regenerating mediators.

Continuous cell separation using dielectrophoresis through asymmetric and periodic microelectrode array.

The study presents a dielectrophoretic cell separation method via three-dimensional (3D) nonuniform electric fields generated by employing a periodic array of discrete but locally asymmetric triangular bottom microelectrodes and a continuous top electrode. Traversing through the microelectrodes, heterogeneous cells are electrically polarized to experience different strengths of positive dielectrophoretic forces, in response to the 3D nonuniform electric fields. The cells that experience stronger positive dielectrophoresis are streamed further in the perpendicular direction to the fluid flow, leaving the cells that experience weak positive dielectrophoresis, which continue to traverse the microelectrode array essentially along the laminar flow streamlines. The proposed method has achieved 87.3% pure live cells harvesting efficiency from a live/dead NIH-3T3 cells mixture, and separation of MG-63 cells from erythrocytes with a separation efficiency of 82.8%. The demonstrated cell separation shows promising applications of the DEP separator for cell separation in a continuous mode.

Perfusion decellularization of porcine esophagus: Study of two processing factors affecting the folded mucosal structure of the esophageal scaffold.

Acellular scaffolds from decellularized donor organs are showing promising clinical results in tissue and organ repair and regeneration. A successful decellularization process is determined by (a) its capability to decellularize complete organs of large animals, (b) retention of the extracellular matrix (ECM) structures and morphologies, and (c) minimal loss of ECM proteins. In this study, porcine esophagi were perfused in full thickness with 0.25% w/v sodium dodecyl sulfate at perfusion rates 0.1-0.2 ml/min for up to 5 days. Decellularized tissues were characterized for their residual DNA, histological staining for their matrix structures, immunohistochemical staining for collagen type IV and laminin, and scanning electron microscopy for structural integrity. Our results showed that full thickness esophageal tissues treated using the horizontal perfusion setup were decellularized with good structural and biochemical integrity in the ECM. Residual DNA content in decellularized tissues was found to be 36 ± 12 ng/mg of tissues (n = 6) which was significantly lower than that of native tissues (p = .00022). Our study showed that the organ must be decellularized in full thickness and perfusion pressure must be controlled to minimize radial expansion. These factors were found to be critical in preserving the folded mucosa in the decellularized tissues.

Esophageal epithelium regeneration on fibronectin grafted poly(L-lactide-co-caprolactone) (PLLC) nanofiber scaffold.

In order to mimic normal epithelium regeneration on synthetic scaffold in vitro, biodegradable elastic poly(l-lactide-co-caprolactone) (PLLC) was processed into nanofibrous scaffold using electrospinning technology. An adhesive protein, fibronectin (Fn), was grafted onto the scaffold fiber surface via a two-step reaction: polyester aminolysis followed by Fn coupling via glutaraldehyde. Tensile testing was performed to measure the effect of aminolysis on the scaffold mechanical properties. The strain decreased but the tensile strength remained almost constant after aminolysis. However, no obvious difference of the nanofiber surface morphology was found after Fn grafting using scanning electron microscopy (SEM). Porcine esophageal epithelial cells were seeded on the Fn bonded scaffold to test the cell growth promotion against the control unmodified PLLC nanofiber scaffold using tissue culture polystyrene (TCPS) plate as a reference. Anti-cytokeratin AE1/AE3 was used as the primary antibody to confirm the esophageal epithelial phenotype. SEM observation, immunostaining and Western Blotting to compare the collagen type IV synthesis showed that the Fn grafted on PLLC scaffold greatly promotes epithelium regeneration. This modified scaffold is expected to be a good candidate for functional esophagus substitutes.

Dual requirements of extracellular matrix protein and chitosan for inducing adhesion contact evolution of esophageal epithelia.

It has been recently shown that chitosan (CHI)/collagen prostheses induced epithelization at the esophagus site of animal model. However, little is known on the biophysical mechanisms of cell adhesion on CHI-based material pertaining to esophagus tissue engineering. In this study, the adhesion contact dynamics of porcine esophageal epithelial cells seeded on CHI surface is probed using confocal-reflectance interference contrast microscopy in conjunction with phase-contrast microscopy. First of all, cells fail to form any adhesion contact on either CHI or elastin (ES)-coated surface. On CHI coated with fibronectin (CHI-FN) or elastin (CHI-ES), strong adhesion contact of cells evolved over time until they reached a steady-state level. The initial cell deformation rates of cells on CHI-FN and CHI-ES are 0.0138 and 0.0151 min(-1), respectively. Interestingly, cells on fibronectin (FN) coated substrate transiently form strong adhesion contact and eventually undergo deadhesion. Moreover, the steady-state adhesion energy of epithelial cells on CHI-FN is 1.73 and 148 times larger than that on CHI-ES and FN, respectively. The actin of cells on CHI-FN transforms from microfilament meshes at cell periphery to stress fibers throughout the cytoplasm during cell seeding. At the same time, vinculin staining demonstrated the evolution of focal adhesion complexes in cells on CHI-FN after 130 min of seeding. Interestingly, CHI-ES induces the formation of focal adhesion complexes in a lesser extent in cell but fails to lead to stress fiber formation. Overall, our study reveals that long-term adhesion contact evolution of esophageal epithelia is only triggered by both extracellular matrix protein and chitosan.

Protein bonding on biodegradable poly(L-lactide-co-caprolactone) membrane for esophageal tissue engineering.

A biodegradable and flexible poly(L-lactide-co-caprolactone) (PLLC) copolymer was synthesized and surface modification has been performed aiming at application as a scaffold in esophageal tissue engineering. The PLLC membrane surface was aminolyzed by 1,6-hexanediamine to introduce free amino groups. Using these amino groups as bridges, fibronectin and collagen were subsequently bonded with glutaraldehyde as a coupling agent. The presence of free amino groups on the aminolyzed PLLC surface was quantified using fluorescamine analysis method, which revealed that the surface NH2 density increased and eventually saturated with increasing 1,6-hexanediamine concentration or reaction time. X-ray photoelectron spectroscopy (XPS) confirmed the presence of both proteins separately on the modified PLLC surface. Water contact angle measurements evaluate the wettability of modified and unmodified PLLC surfaces. Protein-bonded surface presented more hydrophilic and homogeneous, yet PLLC can also adsorb some protein molecules. In vitro long-term (12d) culture of porcine esophageal cells proved that fibronectin- and collagen-modified PLLC surface (denoted PLLC-Fn and PLLC-Col, respectively) can more effectively support the growth of smooth muscle cells and epithelial cells; both modified and unmodified PLLC support fibroblasts growth. Mitochondrial activity assay and cell morphology observation indicate that the PLLC-Fn surface is more favorable to epithelium regeneration than PLLC-Col. These culture results provide much valuable information for our subsequent research on the construction of artificial scaffolds with esophageal function. Fibronectin-integrated PLLC will be a good candidate scaffold to support the growth of all types of esophageal cells.

Characterization, mechanical behavior and in vitro evaluation of a melt-drawn scaffold for esophageal tissue engineering.

Tubular esophageal scaffolds with fiber diameter ranging from 13.9±1.7µm to 65.7±6.2µm were fabricated from the highly elastic poly(l-lactide-co-ε-caprolactone) (PLC) via a melt-drawing method. The morphology, crystallinity, thermal and mechanical properties of the PLC fibers were investigated. They were highly aligned and have a uniform diameter. PLC is found to be semicrystalline consisting of α- and ß- lactide (LA) crystals. The crystallinity increases up to 16.8% with increasing melt-drawing speeds due to strain-induced crystallization. Modulus and strength increases while ductility decreases with an increase in crystallinity of the PLC samples. Moisture will not degrade the overall tensile properties but affect its tangent modulus at the low strain. L929 cells are able to attach and proliferate on the scaffolds very well. The cells seeded on the scaffolds show normal morphology with >90% cell viability after 6 days of culture. These results demonstrate that the PLC fibrous scaffold has good potential for use in esophageal tissue engineering application.

Effect of argon-plasma treatment on proliferation of human-skin-derived fibroblast on chitosan membrane in vitro.

Chitosan is not only a nontoxic, biocompatible, and biodegradable polymer, but has also a chemical structure similar to glycosaminoglycans (GAGs), which promote scarless wound healing of skin. In this study, chitosan membranes were treated with argon plasma to improve their surface hydrophilicity. The results showed that the water contact angles of these surface-treated membranes were significantly reduced from 60.76 to 11.57 degrees . The total surface energy was increased from 41.06 to 67.31 mJ/m(2), with 60-86.95% improvement in the gamma-negative component and a 20% difference in the nonpolar component. Argon-plasma-treated chitosan membranes exhibited excellent attachment, migration, and proliferation of the human-skin-derived fibroblasts (hSFs) compared to the untreated ones. It was found that the duration of argon-plasma treatment influenced the cell proliferation, and the optical densities in MTT assay were enhanced. Argon-plasma treatment improved the surface hydrophilicity of chitosan membranes and promoted the attachment and proliferation of hSFs.

Cryogenic electrospinning: proposed mechanism, process parameters and its use in engineering of bilayered tissue structures.

BACKGROUND: Conventional electrospun scaffolds have very small pores, thus limiting cellular infiltration, tissue ingrowth and vascularization in tissue engineering applications. The cryogenic electrospinning process overcame the small pore size constraints found in conventional electrospun scaffolds. AIM: The aim of this paper is to propose a mechanism for cryogenic electrospinning and how scaffold pore size can be controlled. MATERIALS & METHODS: We studied the roles of ice crystals in controlling the pore size of cryogenic electrospun scaffolds (CES). Based on this understanding, we have successfully fabricated a bilayered scaffold with distinctly different pore sizes. RESULTS: Our study showed that CES pore size was dependent on the structure of the frost layer formed and hence the factors affecting ice deposition. The bilayered scaffold was able to support the coculture of human dermal fibroblasts and keratinocytes. CONCLUSION: The larger pores of CES add versatility to the use of electrospun scaffolds in tissue engineering applications.

Repair of osteochondral defects with rehydrated freeze-dried oligo[poly(ethylene glycol) fumarate] hydrogels seeded with bone marrow mesenchymal stem cells in a porcine model.

Current surgical techniques for osteochondral injuries in young active patients are inadequate clinically. Novel strategies in tissue engineering are continuously explored in this area. Despite numerous animal studies that have shown encouraging results, very few large-scale clinical trials have been done to address this area of interest. To facilitate the eventual translation from rabbit to human subjects, we have performed a study using bone marrow-derived mesenchymal stem cell (BMSC)-oligo[poly(ethylene glycol) fumarate] (OPF) hydrogel scaffold in a porcine model. Our objective was to analyze the morphology of BMSCs seeded into rehydrated freeze-dried OPF hydrogel and in vivo gross morphological and histological outcome of defects implanted with the BMSCs-OPF scaffold in a porcine model. The analyses were based on magnified histologic sections for different types of cartilage repair tissues, the outcome of the subchondral bone, scaffold, and statistical assessment of neotissue-filling percentage, cartilage phenotype, and Wakitani scores. The morphology of the BMSCs seeded into the rehydrated freeze-dried OPF scaffold was observed 24 h after cell seeding, through the phase-contrast microscope. The three-dimensional and cross-sectional structure of the fabrication was analyzed through confocal microscopy and histological methods, respectively. The BMSCs remained viable and were condensed into many pellet-like cell masses with a diameter ranging from 28.5 to 298.4 (113.5±47.9) µm in the OPF scaffold. In vivo osteochondral defect repair was tested in 12 defects created in six 8-month-old Prestige World Genetics micropigs. The implantation of scaffold alone was used for control. Gross morphological, histological, and statistical analyses were performed at 4 months postoperatively. The scaffold-MSC treatment led to 99% defect filling, with 84% hyaline-like cartilage at 4 months, which was significantly (p<0.0001) more than the 54% neotissue filling and 39% hyaline-like cartilage obtained in the scaffold-only group. The implantation of BMSCs in freeze-dried OPF hydrogel scaffold, which created a conducive environment for cell infiltration and clustering, could fully repair chondral defects with hyaline-like cartilage. This protocol provides a clinically feasible procedure for osteochondral defect treatment.

Cryogenic prototyping of chitosan scaffolds with controlled micro and macro architecture and their effect on in vivo neo-vascularization and cellular infiltration.

A major challenge in tissue engineering has been to develop scaffolds with controlled complex geometries, on both the macro- and micro-scale. One group of techniques, using rapid prototyping (RP) processes, has the capability to produce complex three-dimensional structures with good control over the size, geometry, and connectivity of the pores. In this article, a novel technique based on RP technology, that is, cryogenic prototyping (CP), that has the capability to fabricate scaffolds with controlled macro- and micro-structures, is presented. Our in vivo studies showed that the micro architecture (i.e., both pore size and pore orientation) and macro structures of the CP scaffolds affect both cellular infiltration and neo-vascularization. Full cellular infiltration and neo-vascularization were observed after 28 days in scaffolds with micropore sizes of 90 microm. In addition, it was observed that channels (300 microm) created in scaffolds were effective at enhancing cellular infiltration and vascularization. Our results have demonstrated that CP is a viable method for fabricating scaffolds for a wide range of tissue engineering applications.

Fabrication and in vitro and in vivo cell infiltration study of a bilayered cryogenic electrospun poly(D,L-lactide) scaffold.

Cryogenic electrospinning has previously been demonstrated for controlling the pore sizes of electrospun scaffolds, which has been impossible with traditional electrospinning processes. This article describes the application of the cryogenic technique to fabricate a bilayered electrospun poly(D,L-lactide) scaffold (BLES) in a single uninterrupted process. The resulting BLES consisted of a traditional electrospun (ES) fibrous layer with a dense pore area of 17 +/- 3 microm(2) adjacent to a cryogenic electrospun layer (CES) with a pore area of 3300 +/- 500 microm(2). The significance of this bilayered scaffold was to mimic the anatomical structure of tissues with dense basement membrane followed by loose and highly porous connective tissue such as skin and blood vessels. Cell infiltration in the BLES was compared in vitro and in vivo. Both studies suggested the CES supported high cell infiltration, whereas the ES could serve as a physical barrier to prevent cell infiltration across the CES-ES boundary because of its size exclusion. The bilayered structure produced by this technique suggests a great potential for engineering tissues with similar architectures.

In vitro cell infiltration and in vivo cell infiltration and vascularization in a fibrous, highly porous poly(D,L-lactide) scaffold fabricated by cryogenic electrospinning technique.

One of the obstacles limiting the application of electrospun scaffolds for tissue engineering is the nanoscale pores that inhibit cell infiltration. In this article, we describe a technique that uses ice crystals as templates to fabricate cryogenic electrospun scaffolds (CES) with large three-dimensional and interconnected pores using poly(D,L-lactide) (PLA). Manipulating the humidity of the electrospinning environment the pore sizes are controlled. We are able to achieve pore sizes ranging from 900 +/- 100 microm(2) to 5000 +/- 2000 microm(2) depending on the relative humidity used. Our results show that cells infiltrated the CES up to 50 microm in thickness in vitro under static culture conditions whereas cells did not infiltrate the conventional electrospun scaffolds. In vivo studies demonstrated improved cell infiltration and vascularization in the CES compared with conventionally prepared electrospun scaffolds. In gaining control of the pore characteristics, we can then design CES that are optimized for specific tissue engineering applications.

Effect of electrospun poly(D,L-lactide) fibrous scaffold with nanoporous surface on attachment of porcine esophageal epithelial cells and protein adsorption.

Electrospun scaffolds have been increasingly used in tissue engineering applications due to their size-scale similarities with native extracellular matrices. Their inherent fibrous features may be important in promoting cell attachment and proliferation on the scaffolds. In this study, we explore the technique of fabricating electrospun fibers with nano-sized porous surfaces and investigate their effects on the attachment of porcine esophageal epithelial cells (PEECs). Porosity was introduced in electrospun poly(D,L-lactide) fibers by creating vapor-induced phase separation conditions during electrospinning. The nanoporous fiber scaffolds were mechanically weaker than the conventional solid fiber scaffolds and solvent-cast films of the same polymer. However, the nanoporosity of the fibers was found to enhance the levels of adsorbed protein from a dilute solution of fetal bovine serum. The amount of protein adsorbed by nanoporous fiber scaffolds was approximately 80% higher than the solid fiber scaffolds. This corresponds to an estimated 62% increase in surface area of the porous fibers than the solid fibers. By comparison, the solvent-cast films adsorbed low levels of protein from the FBS solution. In addition, the porous fibers were found to be advantageous in enhancing initial cell attachment as compared with the solid fibers and solvent-cast films. It was observed that nanoporous fiber scaffolds seeded with PEECs had significantly greater number of viable cells attached than the solid fiber scaffolds after 10 and 24 h in culture. Hence, our results indicate that nanosized porous surfaces on electrospun fibers enhance both protein adsorption and cell attachment. These findings provide a method to improve cell-matrix interactions of electrospun scaffolds for tissue engineering applications.

Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid).

Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 x 10(-7) J m(-2)) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.