Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial denaturation or contraction, weight loss, and severe calcification (13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0.2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinked sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 weeks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-50%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less than 10% in monolayers and more than 50% in floating collagen gel. A small amount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels.     

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.     

Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.     

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.     

Responses of human keratocytes to micro- and nanostructured substrates

We have previously shown that human corneal epithelial cells respond to synthetic topographic features with dimensions similar to those found in the native human corneal basement membrane. Epithelial cells integrated inputs from substrate topography and soluble factors in the culture medium to generate alignment responses to substrate topographic anisotropies. Human keratocytes are the main cellular components of the stroma, the tissue that underlies the corneal epithelium. Here we report that keratocytes aligned more strongly than epithelial cells along topographic patterns of grooves and ridges. On patterns with pitches of 800 nm and larger approximately 70% of keratocytes were aligned along the patterns compared to 35% for epithelial cells. On 70 nm-wide ridges on a 400-nm pitch, keratocyte alignment dropped to 45%, whereas epithelial cell alignment remained constant. Similarly to epithelial cells, focal adhesions and associated stress fibers in keratocytes were aligned mainly along the substrate topographies, although oblique orientations were also observed. Furthermore, keratocytes cultured on the nanoscale patterns had fewer stress fibers and focal adhesions than cells cultured on microscale patterns or on smooth substrates.     

The scale of substratum topographic features modulates proliferation of corneal epithelial cells and corneal fibroblasts

The cornea is a complex tissue composed of different cell types, including corneal epithelial cells and keratocytes. Each of these cell types are directly exposed to rich nanoscale topography from the basement membrane or surrounding extracellular matrix. Nanoscale topography has been shown to influence cell behaviors, including orientation, alignment, differentiation, migration, and proliferation. We investigated whether proliferation of SV40-transformed human corneal epithelial cells (SV40-HCECs), primary human corneal epithelial cells (HCECs), and primary corneal fibroblasts is influenced by the scale of topographic features of the substratum. Using basement membrane feature sizes as our guide and the known dimensions of collagen fibrils of the corneal stroma (20-60 nm), we fabricated polyurethane molded substrates, which contain anisotropic feature sizes ranging from 200-2000 nm on pitches ranging from 400 to 4000 nm (pitch = ridge width + groove width). The planar regions separating each of the six patterned regions served as control surfaces. Primary corneal and SV40-HCEC proliferation decreased in direct response to decreasing nanoscale topographies down to 200 nm. In contrast to corneal epithelial cells, corneal fibroblasts did not exhibit significantly different response to any of the topographies when compared with planar controls at 5 days. However, decreased proliferation was observed on the smallest feature sizes after 14 days in culture. Results from these experiments are relevant in understanding the potential mechanisms involved in the control of proliferation and differentiation of cells within the cornea.     

Multipotent stem cells in human corneal stroma

Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neurofilament protein, and beta-tubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics.     

IL-4 induces eotaxin production in corneal keratocytes but not in epithelial cells

BACKGROUND: In severe allergic eye diseases, the breakdown of epithelial barrier function can lead to severe corneal damage such as erosions or ulcers which often resist treatment. Although eosinophils are thought to play a crucial role in corneal tissue damage in severe ocular allergy, the mechanisms of eosinophil recruitment to the cornea has not been fully clarified. Eotaxin has been found in tears of severe allergic patients with corneal ulcer. In this study, we investigated whether the Th2 cytokine interleukin-4 (IL-4) induces eotaxin production in human corneal epithelial cells and keratocytes. METHODS: Primary cultures of human corneal epithelial cells and keratocytes were incubated with IL-4 and/or TNF-alpha for 48 h. Released eotaxin was measured by ELISA, and the eotaxin proteins were visualized by immunocytochemistry. Eotaxin mRNA expression in cultured cells was analyzed by RT-PCR. RESULTS: IL-4 induced eotaxin production in keratocytes in a dose- and time-dependent manner which was enhanced by TNF-alpha. There was no detectable eotaxin produced by corneal epithelial cells (<5 pg/ml). The cytoplasm of keratocytes incubated with IL-4 stained positively against anti-eotaxin antibodies, while eotaxin mRNA was detected in keratocytes incubated with IL-4. CONCLUSIONS: Human corneal keratocytes, but not epithelial cells, are capable of producing eotaxin by stimulation with IL-4. Our results suggest that eotaxin production in keratocytes induced by IL-4 may play an important role in eosinophil recruitment to corneal ulcers in allergic ocular disease. Eotaxin production by keratocytes may explain the severity of allergic disease involving the corneal stroma.     

Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges

Various culture systems have been used for examining the anabolic and catabolic functions of isolated chondrocytes as well as for tissue engineering purposes. Perfusion or frequent medium change is beneficial for three-dimensional (3D) cultures of many cell types. In this study, bovine articular chondrocytes (bACs) were grown in 3D collagen sponges with or without medium perfusion (0.33 mL/min) for up to 15 days. The influence of medium perfusion was evaluated using markers of cartilage matrix accumulation, synthesis, and gene expression. Metachromatic matrix, collagen type II, and hyaluronan accumulated around the cells within the collagen sponges. Sulfated glycosaminoglycans (S-GAGs) that accumulated in the sponge exposed to nonperfused control were 130% of that in the perfused sponge at day 7. S-GAG accumulation after 15 days in the nonperfused control was 230% more than at day 7 (p < 0.01). (35)S-sulfate incorporation during the final 18 h of culture in the sponge exposed to nonperfusion was 180% greater than that in the perfused sponge (p < 0.01). Quantitative analyses show that at day 7, aggrecan and collagen type II gene expression were 350% and 240% greater, respectively, in the nonperfused culture than in the perfused one. These results indicate that perfused conditions that are beneficial for other cell types inhibit chondrogenesis by articular chondrocytes in 3D culture.     

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.     

Extracellular Vesicles and Cell–Cell Communication in the Cornea

One question that has intrigued cell biologists for many years is, “How do cells interact to influence one another's activity?” The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100-million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo-like three-dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell–cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Enhanced proliferation and osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with different poly(ethylene terephthalate) fibers

The objective of this study was to investigate the feasibility of collagen sponges mechanically reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers in stem cell culture. A collagen solution with homogeneously dispersed PET fibers was freeze-dried, followed by dehydrothermal cross-linking to obtain the collagen sponge incorporating PET fibers. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 200 microm, irrespective of PET fiber incorporation. As expected, PET fibers incorporation significantly enhanced the compression strength of collagen sponge. When used for rat mesenchymal stem cells (MSC), the collagen sponge incorporating PET fibers was superior to the original collagen sponge without PET fibers incorporation in terms of the initial attachment, proliferation and osteogenic differentiation of cells, irrespective of the amount and diameter of fibers incorporated. The shrinkage of sponges during cell culture was significantly suppressed by the fiber incorporation. It is possible that the shrinkage suppression maintains the three-dimensional inner pore structure of collagen sponges without impairing the cell compatibility, resulting in the superior MSC attachment and the subsequent osteogenic differentiation in the sponge incorporating PET fiber.     

The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells

Corneal stroma is an avascular connective tissue characterized by layers of highly organized parallel collagen fibrils, mono-disperse in diameter with uniform local interfibrillar spacing. Reproducing this level of structure on a nano- and micro-scale may be essential to engineer corneal tissue with strength and transparency similar to that of native cornea. A substrate of aligned poly(ester urethane) urea (PEUU) fibers, 165 ± 55 nm in diameter, induced alignment of cultured human corneal stromal stem cells (hCSSCs) which elaborated a dense collagenous matrix, 8-10 μm in thickness, deposited on the PEUU substratum. This matrix contained collagen fibrils with uniform diameter and regular interfibrillar spacing, exhibiting global parallel alignment similar to that of native stroma. The cells expressed high levels of gene products unique to keratocytes. hCSSCs cultured on PEUU fibers of random orientation or on a cast film of PEUU also differentiated to keratocytes and produced abundant matrix, but lacked matrix organization. These results demonstrate the importance of topographic cues in instructing organization of the transparent connective tissue of the corneal stroma by differentiated keratocytes. This important information will help with design of biomaterials for a bottom-up strategy to bioengineer spatially complex, collagen-based nano-structured constructs for corneal repair and regeneration.     

Comparative phenotypic analysis of articular chondrocytes cultured within type I or type II collagen scaffolds

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.     

体外培养人角膜缘上皮细胞抑制激活态角膜基质细胞的生长

背景：角膜受到损伤后,角膜基质细胞激活转变为成纤维细胞,引起角膜基质瘢痕化,导致视力下降甚至丧失。 目的：观察角膜不同部位上皮细胞与角膜基质细胞的相互作用,探索角膜缘上皮细胞群能否抑制激活态角膜基质细胞的生长。 方法：采用酶消化及机械外力相结合的方法获取人角膜中央、角膜旁中央及角膜缘处角膜上皮细胞与浅层角膜基质细胞,进行体外培养。相差显微镜下观察细胞形态及生长变化。待培养角膜上皮细胞与基质细胞发生接触抑制时,记作“0 周”,采用免疫荧光染色技术检测培养细胞中PCNA及p63蛋白的表达。 结果与结论：培养的角膜上皮细胞与成纤维细胞发生接触抑制时,两种细胞间有明显分界线。角膜缘组上皮细胞中PCNA及p63蛋白均有较高的表达;角膜旁中央组PCNA有较高的表达,p63蛋白阴性表达;角膜中央组PCNA表达较低,p63蛋白阴性表达;从鉴定结果中可以得出只有角膜缘组中存在一定比例的角膜缘上皮干细胞。角膜缘组上皮细胞逐渐包围并化解成纤维细胞,在相互作用4周后,成纤维细胞聚集成死细胞团,缺乏角膜缘干细胞的中央组及旁中央组中成纤维细胞生长面积增加,上皮细胞生长受到抑制甚至死亡。说明体外培养的角膜缘上皮细胞群可以抑制激活态角膜基质细胞的生长。     

A neo-esophagus reconstructed by cultured human esophageal epithelial cells, smooth muscle cells, fibroblasts, and collagen

The scientists involved in this study attempted to develop an artificial esophagus constructed of autologous cells grown by cell culture methods on an extracellular matrix. An artificial esophagus consisting of human esophageal epithelial cells, dermal fibroblasts, and smooth muscle cells isolated from the aortic media, was attempted. The purpose of this study was to examine whether smooth muscle cells could be used in the transforming matrix. Human fibroblasts were embedded in Type I collagen superimposed on the collagen layer of smooth muscle cells. Next, human esophageal epithelial cells were cultured on the collagen layer of the fibroblasts. The resulting collagen sheets were cultured in vitro for 1 week, then transplanted on the latissimus dorsi muscles of athymic rats. The sheets were examined histologically at 1 and 2 weeks using hematoxylin eosin and immunologic stain methods (antiactin antibody). At the end of 2 weeks after transplantation, on microscopic observation of the collagen sheets, it appeared that the epithelial layer, the submucosal tissue layer, and the proper muscle layer had been reconstructed. Additionally, the authors successfully isolated smooth muscle cells from the media of the left gastric artery as a surgical specimen by explant cell culture. The ability to transform collagen sheets consisting of esophageal epithelial cells, fibroblasts, and smooth muscle cells from a surgical specimen into a luminal structure may enable clinical application of the artificial esophagus.     

Perfusion culture enhances osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with poly(glycolic Acid) fiber

The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation.     

Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices

In order to alleviate their extensive contraction, human fibroblast sheets were cultured in combination with three-dimensional matrices (knitted poly(lactic-co-glycolic acid) (PLGA) mesh and collagen-hyaluronic acid (CHA) sponge) to form contiguous dermal constructs for tissue engineering a bilayered skin equivalent. The resulting constructs were viable, and supported the development of bilayered skin equivalents which did not contract over the 4-week culture period. When implanted into full-thickness wounds in nude rats, cultured skin equivalents based on PLGA meshes registered a take rate of 100% and showed an extent of wound contraction that was statistically similar to autografts, while wounds grafted with PLGA meshes without cell sheets contracted more than autografts. On the other hand, skin equivalents based on CHA sponges were all sloughed off within 2 weeks of transplantation. In all cell sheet-incorporated specimens, cells from the constructs infiltrated and produced extracellular matrix within the neo-dermis, shown by positive human leukocyte antigen and collagen I expression. This technique offers an alternative approach for scaffold-based tissue engineering to produce mechanically stable grafts with matured neo-tissue.