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PubMed2006

Diaphragmatic repair through fetal tissue engineering: a comparison between mesenchymal amniocyte- and myoblast-based constructs.

PURPOSE: We have previously shown that fetal tissue engineering is a preferred alternative to diaphragmatic repair in a large animal model. This study was aimed at comparing diaphragmatic constructs seeded with mesenchymal amniocytes and fetal myoblasts in this model. METHODS: Neonatal lambs (n = 14) underwent repair of an experimental diaphragmatic defect with identical scaffolds, either seeded with labeled autologous cells (mesenchymal amniocytes in group 1 and fetal myoblasts in group 2) or as an acellular graft (group 3). At 1 to 12 months postoperatively, implants were harvested for multiple analyses. RESULTS: Repair failure (reherniation or eventration) was significantly higher in group 3 than in groups 1 and 2, with no difference between groups 1 and 2. Seeded fetal myoblasts quickly lost their myogenic phenotype in vivo. All grafts contained cells with a fibroblastic-myofibroblastic profile. Elastin concentrations and both modular and ultimate tensile strengths were significantly higher in group 1 than in groups 2 and 3. There were no differences in glycosaminoglycans and type I collagen levels among the groups. CONCLUSIONS: Diaphragmatic repair with a mesenchymal amniocyte-based engineered tendon leads to improved structural outcomes when compared with equivalent fetal myoblast-based and acellular grafts. The amniotic fluid is a preferred cell source for tissue-engineered diaphragmatic reconstruction.

Animals↗
PubMed2006

Fetal tracheal reconstruction with cartilaginous grafts engineered from mesenchymal amniocytes.

BACKGROUND/PURPOSE: This study was aimed at determining whether cartilaginous grafts engineered from mesenchymal cells normally present in the amniotic fluid could be used in fetal tracheal repair. METHODS: Ovine mesenchymal amniocytes were expanded in culture, labeled with green fluorescent protein, and seeded onto biodegradable scaffold tubes maintained in chondrogenic medium. After chondrogenic differentiation of the constructs was confirmed, they were used to repair either partial or full circumferential tracheal defects in allogeneic fetal lambs (n = 7). Newborns were evaluated for signs of airway compromise. Implants were harvested over a 10-day period postnatally for multiple analyses. RESULTS: All 5 lambs that survived to term were able to breathe spontaneously at birth, 4 (80%) of them without stridor. However, variable degrees of stridor developed over time in all but one animal. Mild-to-moderate tracheal stenosis was present in all specimens. Histologically, grafts contained green fluorescent protein-positive cells, were lined with pseudostratified columnar epithelium, and remodeled into a predominantly fibrous cartilage pattern. Implants showed no significant changes in glycosaminoglycans, collagen, and elastin content at harvest. CONCLUSIONS: Engineered cartilaginous grafts derived from mesenchymal amniocytes may become a viable alternative for tracheal repair. The amniotic fluid can be a practical cell source for engineered tracheal reconstruction.

Amniotic Fluid↗
PubMed2004

Cranial repair using BMP-2 gene engineered bone marrow stromal cells.

BACKGROUND: Bone grafts, allografts, and biocompatible artificial bone substitutes all have their shortcomings when used for the repair of cranial bone defects. Tissue engineered bone shows promise as an alternative for the repair of these defects. MATERIALS AND METHODS: Rabbit bone marrow mesenchymal stromal cells (MSCs) were separated from iliac crest aspirates and expanded in a monolayer culture 1 month before implantation. These MSCs were then infected with replication-defective adenovirus-human BMP-2 genes 1 week before implantation. Bilateral critical-size cranial defects were created in the animal with removal of osteoinductive periosteum and dura. MSCs were mixed with alginate UP (ultrapure) to form MSC/polymer construct. MSCs used for the control site were infected with adenovirus beta-galactosidase (beta-gal). After 1 week, 6 weeks, and 3 months, five rabbits from each experimental group were sacrificed and the cranial defect site was examined by histology study. RESULTS: Near-complete repair of the large size cranial defects using the tissue engineered MSC/alginate construct was observed. The H&E stain and von Kossa's staining should better regenerate bone at the experiment site. A statistically significant difference in bone formation was noted by 3D CT imaging at 3 months post-BMP-2 treatment of the cranial defects (0.79 +/- 0.06 versus 0.47 +/- 0.05 cm(2), P < 0.001) but not at 6 weeks (0.36 +/- 0.04 versus 0.33 +/- 0.03 cm(2), P = 0.347). CONCLUSIONS: Near-complete repair of large cranial defects can be achieved using tissue engineered bone. The use of newly developed polymers as well as the integration of the stem cell concept with gene medicine is necessary to attain this goal.

Adenoviridae↗
PubMed2004

Intestinal tissue engineering: from regenerative medicine to model systems.

BACKGROUND: Tissue engineering is a rapidly emerging field that combines the established disciplines of engineering, biology, and medicine with the goal of fabricating viable tissues and organs. Although initially targeted for applications in regenerative medicine, a novel application of this technology has been to generate experimental model systems for studying biological mechanisms and testing the efficacy of potential therapies. METHODS: Review and analysis of the literature. RESULTS AND CONCLUSION: We present the limitations of existing models and provide examples of how tissue engineering has allowed for the generation of new models that may overcome these limitations.

Animals↗
PubMed2006

Endocrine cell and nerve regeneration in autologous in situ tissue-engineered small intestine.

BACKGROUND: The purpose of this study was to regenerate a larger size of small intestinal tissue than that of our previous study and to evaluate the regeneration of the endocrine cells (ECC) and nerve system of autologous tissue-engineered small intestine. The effect of implantation of large numbers of smooth muscle cells (SMC) for the regeneration of small intestine was also investigated. METHODS: Two types of scaffolds with different cell densities were fabricated: low density (LD) of SMC in the scaffold and high density (HD) of SMC in the scaffold. Both scaffolds were implanted into defects of isolated ileum in a canine model. Animals were sacrificed at 8, 12, 18, and 24 weeks. RESULTS: The area of engineered small intestine in the HD group was four times larger than that in the LD group, although that was smaller in size than the original size of the defect. There were no significant changes in the thickness of regenerated smooth muscle layer (SML) in the LD and HD groups. The numbers of endocrine cells gradually increased after implantation. At 18 weeks of regeneration, the number of ECC reached levels comparable to that of normal mucosa. The nerve fibers extended to the center of the graft area and were observed in regenerated SML and regenerated villi at 24 weeks. CONCLUSIONS: The ECC and nerve fibers were regenerated in autologous in situ tissue-engineered small intestine. Seeding a large number of SMC was not sufficient for the regeneration of the small intestine in a tubular configuration.

Animals↗
PubMed2004

Decellularized vein as a potential scaffold for vascular tissue engineering.

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.

Antigens, Surface↗
PubMed2004

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.

Animals↗
PubMed2006

Schwarz meets Schwann: design and fabrication of biomorphic and durataxic tissue engineering scaffolds.

Tissue engineering is a discipline at the leading edge of the field of computer assisted intervention. This multidisciplinary engineering science attempts to meet the reparative and regenerative needs of tissues and organs based on the notion of design and fabrication of scaffolds- porous, three-dimensional "trellis-like" biomimetic structures that, on implantation, provide a viable environment to recuperate and regenerate damaged cells. Existing scaffold fabrication strategies produce sub-optimal porous labyrinths with contra-naturam straight edges. The biomorphic geometry that mimics the secundam-naturam substrate would be one that is continuous through all space, partitioned into two not-necessarily-equal sub-spaces by a non-intersecting, two-sided surface. Minimal surface geometry is not only ideal to describe such a space but is also the preferentially assumed geometry in natural and pathological or manipulated cells. We present results on the premier attempt in computer-controlled fabrication, modulation, and mechanical characterization of tissue engineering scaffolds based on triply periodic minimal surfaces (TPMS). We also present novel strategies to realize coterminous seeding-feeding networks thereby guaranteeing blood/nutrient supply to the proliferating cells at close proximity. This initiative of linking Schwann's 1838 cell theory with Schwarz's 1865 discovery of TPMS is a significant step to fabricate the previously elusive optimal biomorphic tissue analogs.

Animals↗
PubMed2006

Stem cells: new cell source for myocardial constructs tissue engineering.

Cardiovascular diseases like myocardial infarction, complex congenital heart disease, and subsequent heart failure are a leading cause of morbidity and mortality. Recent advances in tissue engineering arise to address the lack of available tissues and organs for transplantation because cells alone are not capable of recreating complex tissues upon transplantation. Consequently, a very promising approach to repair large scar areas and congenital heart defects may be the use of tissue engineering, in which cells are seeded in three-dimensional matrices of biodegradable polymers to form myocardial constructs. In recent years, there has been a tremendous increase in the understanding of stem cell biology. Stem cells have clonogenic and self-renewing capabilities, and under certain conditions, can differentiate into multiple cell lineages. Recent studies have shown that stem cells can be isolated from a wide variety of tissues, including bone marrow, peripheral blood, muscle, and adipose tissue. We hypothesize that tissue-engineered myocardial constructs with stem cells may fulfill the requirements of native heart muscle and, in the long run, may allow replacement of the injured heart and repair of congenital cardiac defects possible.

Animals↗
PubMed2026

Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.

Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.

Animals↗
PubMed2006

Postnatal myocardial augmentation with skeletal myoblast-based fetal tissue engineering.

BACKGROUND: Cardiac anomalies constitute the most common birth defects, many of which involve variable myocardial deficiencies. Therapeutic options for structural myocardial repair remain limited in the neonatal population. This study was aimed at determining whether engineered fetal muscle constructs undergo milieu-dependent transdifferentiation after cardiac implantation, thus becoming a potential means to increase/support myocardial mass after birth. METHODS: Myoblasts were isolated from skeletal muscle specimens harvested from fetal lambs, labeled by transduction with a retrovirus-expressing green fluorescent protein, expanded in vitro, and then seeded onto collagen hydrogels. After birth, animals underwent autologous implantation of the engineered constructs (n = 8) onto the myocardium as an onlay patch. Between 4 and 30 weeks postoperatively, implants were harvested for multiple analyses. RESULTS: Fetal and postnatal survival rates were 89% and 100%, respectively. Labeled cells were identified within the implants at all time points by immunohistochemical staining for green fluorescent protein. At 24 and 30 weeks postimplantation, donor cells double-stained for green fluorescent protein and Troponin I, while losing skeletal (type II) myosin expression. CONCLUSIONS: Fetal skeletal myoblasts engraft in native myocardium up to 30 weeks after postnatal, autologous implantation as components of engineered onlay patches. These cells also display evidence of time-dependent transdifferentiation toward a cardiomyocyte-like lineage. Further analysis of fetal skeletal myoblast-based constructs for the repair of congenital myocardial defects is warranted.

Animals↗
PubMed2005

Engineering regulatory RNAs.

RNA has long been a favoured medium for in vitro evolution and engineering. Functional RNAs produced in vitro can bind small molecules (aptamers), possess catalytic activity (ribozymes) or do both (aptazymes). A plethora of recent work has shown similar strategies used naturally for gene regulation in bacteria. Interest in these natural systems has inspired an effort to engineer and evolve this activity in vivo. A recent paper by Isaacs et al. describes the engineering and in vivo activity of a small RNA that removes translation inhibition by binding the 5' untranslated region of its target mRNA and making the ribosome-binding site accessible.

Animals↗
PubMed2005

Advancing vascular tissue engineering: the role of stem cell technology.

Atherosclerosis and heart disease are still the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts but the patency of such grafts is limited compared to natural materials. Tissue engineering, whereby living tissue replacements can be constructed, has emerged as a solution to some of these difficulties. This, in turn, is limited by the availability of suitable cells from which to construct the vessels. The development of prosthesis using progenitor cells and switching these into endothelial cells is an important and exciting advance in the field of tissue engineering. Here, we describe recent developments in the use of stem cells for the development of replacement vessels. These paradigm shifts in vascular engineering now offer a new route for effective clinical therapy.

Animals↗
PubMed2004

Assessment of tissue-engineered stomach derived from isolated epithelium organoid units.

OBJECTIVE: Isolated stomach epithelial organoid units developed on biodegradable polymers were transplanted to assess the feasibility of a tissue-engineered stomach. BACKGROUND: Despite recent advances in reconstruction techniques, total gastrectomy is still accompanied by various complications. An alternative treatment would be a tissue-engineered stomach, which replaces the mechanical and metabolic functions of a normal stomach. METHODS: Stomach epithelial organoid units isolated from neonatal rats were seeded onto biodegradable polymers. The constructs implanted into the omenta of adult rats were harvested for examination at designated times. Nine rats underwent a second operation for anastomosis. RESULTS: The constructs resulted in cyst-like formations showing vascularized tissue with neomucosa lining the lumen. The surface morphology as assessed using scanning electron microscopy was similar to that of a native stomach. Immunohistochemical staining for alpha-actin smooth muscle and gastric mucin indicated the presence of a smooth muscle layer and a well-developed gastric epithelium, respectively. The luminal surface of the anastomosed tissue-engineered stomach was well-covered with epithelium. CONCLUSIONS: Epithelium-derived stomach organoid units seeded on biodegradable polymers and transplanted into donor rats were shown to vascularize, survive, and regenerate into complex tissue resembling native stomach. Anastomosis between the units and native small intestine may have the potential to stimulate epithelial growth. This research may provide insight into new approaches to alleviate complications following total gastrectomy.

Animals↗
PubMedDate not supplied

Vitrification of tissue engineered pancreatic substitute.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

Animals↗
PubMed2005

Stem cells, tissue engineering and organogenesis in transplantation.

Tissue engineering is an attempt to generate living tissues for surgical transplantation. In vitro and in vivo approaches have led to the production of vascular and cardiovascular components, bones, cartilages and gastrointestinal tissues. Organogenesis has a different aim, which is to create transplantable organs from embryonic tissue implanted into the recipient's omentum. This approach has been successful in creating kidneys and pancreases in animals. The use of stem cells in organogenesis and in tissue engineering has vastly enlarged the potential for clinical applications. The technique of nuclear transfer offers the possibility of creating cells, which are genetically identical to the host. Tissue engineering and organogenesis represent the future of transplantation in medicine. The progress in this field is of tremendous importance because it can produce a new generation of morphologically complex tissues and organs. In this review, the most relevant experiences in this area are summarized, including its perspectives for therapeutical applications.

Animals↗
PubMed2005

A comparative qualitative histological analysis of tissue-engineered bone using bone marrow mesenchymal stem cells, alveolar bone cells, and periosteal cells.

For tissue-engineered bone formation, autogenous osteogenic cells are of paramount importance for successful bone formation. In order to investigate the donor cell-related differences in tissue-engineered bone, cultured bone marrow mesenchymal stem cells, cultured alveolar bone cells, and cultured periosteal cells were examined for their in vivo potential to form bone. These cells were isolated from dogs, expanded in vitro, mixed with autologous fibrin glue and BMP-2, and then injected into the subcutaneous space on the dorsum of nude mice. Bone formation was evaluated at 12 weeks. Histomorphometric analysis demonstrated that the subcutaneous nodules formed in nude mice contained 26.9% newly formed bone when using the bone marrow mesenchymal stem cells, 41.1% newly formed bone when using the alveolar bone cells, and 58.2% newly formed bone when using the periosteal cells. The results suggest that periosteal cells are the best choice for enhancing bone formation in tissue engineering of bone regeneration.

Alveolar Process↗
PubMed2006

Bone regeneration after enucleation of mandibular cysts: comparing autogenous grafts from tissue-engineered bone and iliac bone.

OBJECTIVE: The aim of this study was to compare bone regeneration after grafting enucleated mandibular cyst cavities using either autogenous osteoblasts cultured on a biomaterial or autogenous spongiose iliac bone. STUDY DESIGN: Twenty patients with 22 mandibular cysts were assessed. Eleven cysts were filled in with tissue-engineered bone (autogenous osteblasts cultured on demineralized bone matrix Osteovit) and 11 with spongiose iliac bone as controls. Panoramic radiographs were taken preoperatively, immediately postoperatively, and 3, 6, and 12 months after surgery. Radiolucency was computer analyzed using gray-level histograms. RESULTS: In both groups bone regeneration took place in a similar fashion. After 3 and 6 months there were few differences in bone density between the groups. However, in radiographic controls after 12 months ossification was considerably stronger in cysts grafted with tissue-engineered bone. CONCLUSION: These results advocate for the clinical application of tissue-engineered bone as an alternative viable filling material for cysts.

Adolescent↗