Tissue-engineered Grafts Research Articles

Repairing the osteochondral defect in goat with the tissue-engineered osteochondral graft preconstructed in a double-chamber stirring bioreactor.

To investigate the reparative efficacy of tissue-engineered osteochondral (TEO) graft for repairing the osteochondral defect in goat, we designed a double-chamber stirring bioreactor to construct the bone and cartilage composites simultaneously in one β-TCP scaffold and observed the reparative effect in vivo. The osteochondral defects were created in goats and all the animals were divided into 3 groups randomly. In groups A, the defect was treated with the TEO which was cultured with mechanical stimulation of stir; in group B, the defect was treated with TEO which was cultured without mechanical stimulation of stir; in groups C, the defect was treated without TEO. At 12 weeks and 24 weeks after operation, the reparative effects in different groups were assessed and compared. The results indicated that the reparative effect of the TEO cultured in the bioreactor was better than the control group, and mechanical stimulation of stir could further improve the reparative effect. We provided a feasible and effective method to construct the TEO for treatment of osteochondral defect using autologous BMSCs and the double-chamber bioreactor.

Training stem cells for treatment of malignant brain tumors.

The treatment of malignant brain tumors remains a challenge. Stem cell technology has been applied in the treatment of brain tumors largely because of the ability of some stem cells to infiltrate into regions within the brain where tumor cells migrate as shown in preclinical studies. However, not all of these efforts can translate in the effective treatment that improves the quality of life for patients. Here, we perform a literature review to identify the problems in the field. Given the lack of efficacy of most stem cell-based agents used in the treatment of malignant brain tumors, we found that stem cell distribution (i.e., only a fraction of stem cells applied capable of targeting tumors) are among the limiting factors. We provide guidelines for potential improvements in stem cell distribution. Specifically, we use an engineered tissue graft platform that replicates the in vivo microenvironment, and provide our data to validate that this culture platform is viable for producing stem cells that have better stem cell distribution than with the Petri dish culture system.

Bioartificial fabrication of regenerating blood vessel substitutes: requirements and current strategies

This work reviews the tremendous development in the field of vascular graft tissue engineering driven by a clear and increasing clinical need for functional vascular replacements able to grow and remodel. The different strategies to tissue engineer blood vessels are presented, from the classical approach of a living implant generated in vitro by conditioning a cell-seeded scaffold to remarkable paradigm shifts either i) toward a completely biology-driven strategy (scaffold-free approaches) or ii) the opposite tendency of cell-free scaffolds aiming at eliciting the host reaction for in situ tissue engineering. In the scaffold-based approaches emphasis is given to the material choice.

New perspectives for articular cartilage repair treatment through tissue engineering: A contemporary review.

In this paper review we describe benefits and disadvantages of the established methods of cartilage regeneration that seem to have a better long-term effectiveness. We illustrated the anatomical aspect of the knee joint cartilage, the current state of cartilage tissue engineering, through mesenchymal stem cells and biomaterials, and in conclusion we provide a short overview on the rehabilitation after articular cartilage repair procedures. Adult articular cartilage has low capacity to repair itself, and thus even minor injuries may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. Numerous efforts have been made to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, and to date several researchers aim to implement clinical application of cell-based therapies for cartilage repair. A literature review was conducted on PubMed, Scopus and Google Scholar using appropriate keywords, examining the current literature on the well-known tissue engineering methods for the treatment of knee osteoarthritis.

Translational Research: The CD34+ Cell Is Crucial for Large-Volume Bone Regeneration from the Milieu of Bone Marrow Progenitor Cells in Craniomandibular Reconstruction

This study investigated the role of the bone marrow-derived CD34+ cell in a milieu of osteoprogenitor cells, bone marrow plasma cell adhesion molecules, recombinant human bone morphogenetic protein (rhBMP), and a matrix of crushed cancellous allogeneic bone in the clinical regeneration of functionally useful bone in craniomandibular reconstructions. The history and current concepts of bone marrow hematopoietic stem cells and mesenchymal stem cells are reviewed as they relate to bone regeneration in large continuity defects of the mandible. Patients with 6- to 8-cm continuity defects of the mandible with retained proximal and distal segments were randomized into two groups. Group A received an in situ tissue-engineered graft containing 54 ± 38 CD34+ cells/mL along with 54 ± 38 CD44+, CD90+, and CD105+ cells/mL together with rhBMP-2 in an absorbable collagen sponge (1 mg/cm of defect) and crushed cancellous allogeneic bone. Group B received the same graft, except the CD34+ cell concentration was 1,012 ± 752 cells/mL. The results were analyzed clinically, radiographic bone density was measured in Hounsfield units (HU), and specimens were analyzed histomorphometrically. Forty patients participated (22 men and 12 women; mean age, 57 years). Eight of 20 group A patients (40%) achieved the primary endpoint of mature bone regeneration, whereas all 20 group B patients (100%) achieved the primary endpoint. CD34+ cell counts above 200/mL were associated with achievement of the primary endpoint. Bone density was lower in group A (424 ± 115 HU) than in group B (731 ± 98 HU). Group A bone showed a mean trabecular bone area of 36% ± 10%, versus 67% ± 13% for group B. The CD34+ cell functions as a central signaling cell to mesenchymal stem cells and osteoprogenitor cells in bone regeneration. The mechanism of bone marrow-supported grafts requires a complete milieu to regenerate large quantities of functionally useful bone. CD34+ cell counts in a concentration of at least 200/mL in composite grafts are directly correlated to clinically successful bone regeneration.

Tissue-engineered dermo-epidermal skin analogs exhibit de novo formation of a near natural neurovascular link 10 weeks after transplantation

Human autologous tissue-engineered skin grafts are a promising way to cover skin defects. Clearly, it is mandatory to study essential biological dynamics after transplantation, including reinnervation. Previously, we have already shown that human tissue-engineered skin analogs are reinnervated by host nerve fibers as early as 8 weeks after transplantation. In this study, we tested the hypothesis that there is a de novo formation of a "classical" neurovascular link in tissue-engineered and then transplanted skin substitutes. Keratinocytes, melanocytes, and fibroblasts were isolated from human skin biopsies. After expansion in culture, keratinocytes and melanocytes were seeded on dermal fibroblast-containing collagen type I hydrogels. These human tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds on the back of immuno-incompetent rats. Grafts were analyzed after 3 and 10 weeks. Histological sections were examined with regard to the ingrowth pattern of myelinated and unmyelinated nerve fibers into the skin analogs using markers such as PGP9.5, NF-200, and NF-160. Blood vessels were identified with CD31, lymphatic vessels with Lyve1. In particular, we focused on alignment patterns between nerve fibers and either blood and/or lymphatic vessels with regard to neurovascular link formation. 3 weeks after transplantation, blood vessels, but no nerve fibers or lymphatic vessels could be observed. 10 weeks after transplantation, we could detect an ingrowth of myelinated and unmyelinated nerve fibers into the skin analogs. Nerve fibers were found in close proximity to CD31-positive blood vessels, but not alongside Lyve1-positive lymphatic vessels. These data suggest that host-derived innervation of tissue-engineered dermo-epidermal skin analogs is initiated by and guided alongside blood vessels present early post-transplantation. This observation is consistent with the concept of a cross talk between neurovascular structures, known as the neurovascular link.

Design of 3D Hybrid Composite Scaffolds: Effect of Composition on Scaffold Structure and Cell Proliferation

SummaryThe aim of this study was to develop multi‐phase composite 3D scaffolds and to investigate the in vitro degradation performance, the cell seeding capacity and cells proliferation onto/into the composite scaffolds. Multi‐phase composite scaffolds were formulated by solvent casting and particulate leaching technique, hydroxyapatite (HAP) was used to improve the mechanical properties of poly[(DL‐lactide)‐coglycolide] (PLGA) polymer scaffold, while chitosan (CTS) was added to the formulation for its bioactivity, osteoconductivity and bioadhesive properties. The in vitro degradation results demonstrated that the composite scaffold had a degradation rate correlating with their composition and structural features. Adult fibroblast cells were seeded on the surface of the composite scaffolds. Cell viability and long‐term proliferation onto/into composite scaffolds were evaluated. The results showed that viable cells attached on the surface of scaffolds gradually migrated into the porous scaffold. These results suggest that the two‐phase scaffold, namely PLGA/HAP and PLGA/CTS composite scaffolds, are promising grafts for bone tissue engineering. Further studies are in progress to design an improved three‐phase composite construct obtained combining PLGA, HAP and CTS in a wafer‐like scaffold.

Stem cells in vascular graft tissue engineering for congenital heart surgery

Many children with congenital heart disease need prosthetic replacement grafts in the form of new valves, conduits and patches. Owing to the lack of growth potential of the currently available grafts, a child born with congenital heart defects may typically need to undergo major cardiac surgery several times throughout their lifetime. A very promising approach to solve this problem may be the use of tissue engineering, in which stem cells are seeded to form living-tissue products that have structural and functional properties that can be used to restore, maintain or improve tissue function, and help to building the ‘ideal valve and patch’ to be used in congenital heart surgery. In this review, we will summarize the current knowledge and scientific progress in the field of vascular tissue engineering and stem cell differentiation with specific application to congenital heart surgery.

Acellular matrix as a substrate for tissue-engineered graft of heart valve

In many cases heart valve prosthetics is the only solution to save patient’s life. All mechanical prosthetics currently used are not able to perform function in the body fully because non-living materials are used for their production. Tissue engineering provides the reconstruction of viable valves using stem cells. Acellularized three-dimensional tissue scaffolds as a matrix for autologous cells do improve function of heart valves and promote heart regeneration.

Co-Culture of Human Adipose-Derived Stem Cells with Tenocytes Increases Proliferation and Induces Differentiation into a Tenogenic Lineage

Seeding acellularized tendons with cells is an approach for creating tissue-engineered tendon grafts with favorable biomechanical properties. It was the authors' aim to evaluate whether human adipose-derived stem cells could replace tenocytes for scaffold seeding. Adipose-derived stem cells and tenocytes were co-cultured in different ratios (3:1, 1:1, and 1:3) and with three different methods: (1) direct co-culture, (2) tenocyte-conditioned media on adipose-derived stem cells, and (3) an insert system to keep both cell types in the same media without contact. Proliferation, collagen production, and tenogenic marker expression were measured by hematocytometry, immunocytochemistry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. Proliferation and collagen production were similar for tenocytes and adipose-derived stem cells alone. Phenotype difference between adipose-derived stem cells and tenocytes was indicated by higher tenascin C and scleraxis expression in tenocytes. Proliferation was increased in direct co-cultures, especially at an adipose-derived stem cells-to-tenocyte ratio of 3:1, and for tenocytes in adipose-derived stem cell-conditioned media. Direct co-culture caused significant up-regulation in tenascin C expression in adipose-derived stem cells (4.0-fold; p<005). In tenocyte-conditioned media, tenascin C expression was up-regulated 2.5-fold (p<0.05). In the insert system, tenascin C expression was up-regulated 2.3-fold (p<0.05). Adipose-derived stem cells are good candidates for tendon tissue engineering because they are similar to tenocytes in proliferation and collagen production. With an optimal ratio of 3:1, they increase proliferation in co-culture and change their phenotype toward a tenogenic direction.

Urethral Reconstruction Using Bone Marrow Mesenchymal Stem Cell– and Smooth Muscle Cell–Seeded Bladder Acellular Matrix

BackgroundCongenital or acquired abnormalities may lead to a urethral defect that often requires surgical reconstruction. The traditional methods often lead to complications, including urethrocutaneous fistulae and strictures. In this study, we proposed to construct a tissue-engineered sheet graft (TESG) using a bone marrow mesenchymal stem cell (BMSC)– and smooth muscle cell (SMC)–seeded bladder acellular matrix (BAM) for urethral reconstruction. MethodsRabbit BMSCs and SMCs were isolated, expanded, and identified in vitro before seeding into BAM as the experimental group, whereas BAM-only was the control group. The graft was used to construct TESG for implantation into the rabbit omentum for 2 weeks before urethral reconstruction. We divided 24 male rabbits into four experimental groups six each, and six other were the control group. Histological analysis was performed at 2 weeks, 4 weeks, 8 weeks, and 16 weeks postoperatively. Retrograde urethrography was performed at 16 weeks postoperatively. ResultsAll experimental rabbits survived to they were humanly killed. At 8 weeks, there was no difference between the graft and the normal urethra with no severe shrinkage. At 8 and 16 weeks after TESG grafting in vivo, multilayer urothelium covered the graft, neovascularization was visible within the center of TESG, and organized smooth muscle bundles were present. Retrograde urethrography failed to demonstrate diverticula formation or urethral stricture. Three control rabbits died within 4 weeks postoperatively. Autopsy showed their urethras to be almost completely blocked whereas another three hosts displays urethral strictures. ConclusionA TESG was constructed using a BMSC- and SMC-seeded BAM for urethral reconstruction.

In Vitro Biocompatibility and Antibacterial Efficacy of a Degradable Poly(l-lactide-co-epsilon-caprolactone) Copolymer Incorporated with Silver Nanoparticles

Silver nanoparticles (Ag-nps) are currently used as a natural biocide to prevent undesired bacterial growth in clothing, cosmetics and medical products. The objective of the study was to impart antibacterial properties through the incorporation of Ag-nps at increasing concentrations to electrospun degradable 50:50 poly(L-lactide-co-epsilon-caprolactone) scaffolds for skin tissue engineering applications. The biocompatibility of the scaffolds containing Ag-nps was evaluated with human epidermal keratinocytes (HEK); cell viability and proliferation were evaluated using Live/Dead and alamarBlue viability assays following 7 and 14days of cell culture on the scaffolds. Significant decreases in cell viability and proliferation were noted for the 1.0mg(Ag) g(scaffold)(-1) after 7 and 14days on Ag-nps scaffolds. After 14days, scanning electron microscopy revealed a confluent layer of HEK on the surface of the 0.0 and 0.1mg(Ag) g(scaffold)(-1). Both 0.5 and 1.0mg(Ag) g(scaffold)(-1) were capable of inhibiting both Gram positive and negative bacterial strains. Uniaxial tensile tests revealed a significant (p<0.001) decrease in the modulus of elasticity following Ag-nps incorporation compared to control. These findings suggest that a scaffold containing between 0.5 and 1.0mg(Ag) g(scaffold)(-1) is both biocompatible and antibacterial, and is suitable for skin tissue engineering graft scaffolds.

Vascular tissue engineering: from in vitro to in situ

Blood vessels transport blood to deliver oxygen and nutrients. Vascular diseases such as atherosclerosis may result in obstruction of blood vessels and tissue ischemia. These conditions require blood vessel replacement to restore blood flow at the macrocirculatory level, and angiogenesis is critical for tissue regeneration and remodeling at the microcirculatory level. Vascular tissue engineering has focused on addressing these two major challenges. We provide a systematic review on various approaches for vascular graft tissue engineering. To create blood vessel substitutes, bioengineers and clinicians have explored technologies in cell engineering, materials science, stem cell biology, and medicine. The scaffolds for vascular grafts can be made from native matrix, synthetic polymers, or other biological materials. Besides endothelial cells, smooth muscle cells, and fibroblasts, expandable cells types such as adult stem cells, pluripotent stem cells, and reprogrammed cells have also been used for vascular tissue engineering. Cell-seeded functional tissue-engineered vascular grafts can be constructed in bioreactors in vitro. Alternatively, an autologous vascular graft can be generated in vivo by harvesting the capsule layer formed around a rod implanted in soft tissues. To overcome the scalability issue and make the grafts available off-the-shelf, nonthrombogenic vascular grafts have been engineered that rely on the host cells to regenerate blood vessels in situ. The rapid progress in the field of vascular tissue engineering has led to exciting preclinical and clinical trials. The advancement of micro-/nanotechnology and stem cell engineering, together with in-depth understanding of vascular regeneration mechanisms, will enable the development of new strategies for innovative therapies.

Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.

SLIT3–ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo

Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called “pericytic” MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, “non-pericytic” MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications.

Implantation of a novel tissue-engineered graft in a large tendon defect initiated inflammation, accelerated fibroplasia and improved remodeling of the new Achilles tendon: a comprehensive detailed study with new insights

We constructed a new artificial collagen-based graft as a tendon proper and covered it with a polydioxanone sheath. This bioimplant was tested in vitro and also its effectiveness was tested in a large tendon defect model in vivo. A 2-cm full defect in the left Achilles tendon of all animals (n = 120) was created. The animals were andomly divided into three groups: control (n = 40), treated with collagen-based graft (n = 40) and treated with collagen-Polydioxanone-based graft (n = 40). Clinical examination was done weekly. The animals were euthanized at 60 and 120days post-injury (DPI). The serum level of platelet-derived growth factor (PDGF) was measured. Hydroxyproline and dry matter content together with gross morphologic, histomorphometric, ultrastructural and biomechanical characteristics of the healing tissues were studied. The mechanism of host-graft interactions was studied in another 55 pilot animals. The graft was able to initiate inflammation, accelerate fibroplasia and improve remodeling of the neotenon in the defect area. Except for small remnants, most parts of the implants were gradually absorbed and substituted by a newly regenerated tendon. The preserved remnants were accepted as a part of neotenon and acted as scaffolds for the newly regenerated collagen fibrils. Unlike the controls, the treated lesions showed lower peritendinous adhesion, muscle fibrosis and atrophy and higher hydroxyproline concentration, dry matter content, ultimate strength, yield strength and modulus of elasticity. Numbers, diameter, density and differentiation of collagen fibrils were also greater in the treated lesions than the control ones. This study showed that the implant was cytocompatible, biodegradable, biocompatible and effective in tendon healing.

Biofabrication under fluorocarbon: a novel freeform fabrication technique to generate high aspect ratio tissue-engineered constructs.

Bioprinting is a recent development in tissue engineering, which applies rapid prototyping techniques to generate complex living tissues. Typically, cell-containing hydrogels are dispensed layer-by-layer according to a computer-generated three-dimensional model. The lack of mechanical stability of printed hydrogels hinders the fabrication of high aspect ratio constructs. Here we present submerged bioprinting, a novel technique for freeform fabrication of hydrogels in liquid fluorocarbon. The high buoyant density of fluorocarbons supports soft hydrogels by floating. Hydrogel constructs of up to 30-mm height were generated. Using 3% (w/v) agarose as the hydrogel and disposable syringe needles as nozzles, the printer produced features down to 570-μm diameter with a lateral dispensing accuracy of 89 μm. We printed thin-walled hydrogel cylinders measuring 4.8 mm in height, with an inner diameter of ∼2.9 mm and a minimal wall thickness of ∼650 μm. The technique was successfully applied in printing a model of an arterial bifurcation. We extruded under fluorocarbon, cellularized alginate tubes with 5-mm outer diameter and 3-cm length. Cells grew vigorously and formed clonal colonies within the 7-day culture period. Submerged bioprinting thus seems particularly suited to fabricate hollow structures with a high aspect ratio like vascular grafts for cardiovascular tissue engineering as well as branching or cantilever-like structures, obviating the need for a solid support beneath the overhanging protrusions.

Induction of Angiogenesis and Vasculogenesis in Cell‐Embedded Biomaterials

AbstractThe central role of prevascularization of engineered tissue grafts in postimplantational survival and integration is becoming increasingly appreciated. An in‐depth understanding of the regulating factors and intricacies of generation of three‐dimensional vascular networks in vitro will facilitate effective fabrication of clinically relevant vascularized tissues. In this review we aim to examine the influence of different biomaterials on vasculogenesis and angiogenesis, with particular focus on the impact of various matrix properties, such as composition, stiffness and geometry, on the resulting vasculature. Additionally, the contribution of externally applied mechanical forces, mimicking blood flow patterns, to tissue vascularization efforts is reviewed. We present here pivotal studies focusing on the influence of mechanical forces, such as shear stress and stretching tension, on vascular network formation in biomaterial‐based scaffolds. Comprehensive understanding of the key factors dictating the patterns and functionality of engineered vasculature will facilitate more efficient fabrication of viable tissue grafts, with a broad range of medical applications.

Peeling model for cell adhesion on electrospun polymer nanofibres

Need for controlled drug delivery has gained immense attention over the conventional dosage forms due to improved therapeutic efficacy and reduced toxicity by controlled delivery. Recently, electrospun fibres have gained wide attention particularly for drug delivery systems, tissue engineering and vascular grafts for biomedical application. In the present work, the feasibility of producing fibrous composite of polylactic acid (PLA) and hydroxyapatite with silver nitrate using electrospinning technique was explored to understand the antibacterial property of the dressing. These non-woven fibres were post processed and heat treated with UV radiations. Such heat treatment is known to reduce the ionic silver to silver nanoparticles and also improve the crystalline properties of hydroxyapatite and PLA. Antimicrobial tests show that these fibres have maintained antibacterial properties against Staphylococcos aureus. It was noticed that there was no discolouration in the wound mat. To test the biocompatibility of these fabrics, the electrospun mats were cultured with fibroblasts. The results indicated that the cells attached and proliferated as continuous layers and maintained a healthy morphology. Once the biomaterial is implanted, no control is possible either over the biomaterial characteristics or the healing process. At this point the modelling process takes over. The present study is extended to a mathematical model for a primary step in tissue regeneration–cell adhesion, using a ‘one-dimensional peeling model’.

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma.

To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology. Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy. HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay. Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.