Tissue-engineered Grafts Research Articles

Fast-degrading elastomer enables rapid remodeling of a cell-free synthetic graft into a neoartery

Host remodeling is important for the success of medical implants including vascular substitutes. Synthetic and tissue-engineered grafts have yet to show clinical effectiveness in arteries smaller than 5 mm. We designed cell-free biodegradable elastomeric grafts that degrade rapidly to yield neo-arteries nearly free of foreign materials 3 months after interposition grafting in rat abdominal aorta. This design focuses on enabling rapid host remodeling. Three months post-implantation, the neo-arteries resemble native arteries in the following aspects: regular, strong and synchronous pulsation, a confluent endothelium and contractile smooth muscle layers, co-expression of elastin, collagen and glycosaminoglycan, and tough and compliant mechanical properties. Therefore, future study employing large animal models more representative of human vascular regeneration is warranted before clinical translation. This cell-free approach represents a philosophical shift from the prevailing focus on cells in vascular tissue engineering, and may impact regenerative medicine in general.

The influence of substrate stiffness on the behavior and functions of Schwann cells in culture

Solid tissues in the body possess a range of stiffness and provide cells with an instructive microenvironment. Scaffolds in tissue engineering should be rationally designed to become an adhesion substrate friendly to cells. Schwann cells are the principal glial cell in the peripheral nervous system and used as support cells for generating tissue-engineered nerve grafts. Although an important mechanical cue, substrate stiffness, has been documented to make significant effects on many types of cells cultured on the substrate, such a study for Schwann cells is still lacking. In this study, we investigated cell adhesion, survival, proliferation, migration, cytoskeleton, and neurotrophic actions of Schwann cells cultured on polyacrylamide gel substrates with different stiffness, and determined an optimal elastic modulus value for these substrates. Our data not only highlight the importance of substrate stiffness in the crosstalk between Schwann cells and surrounding microenvironment, but also introduce a new parameter, in addition to biocompatibility, biodegradability, and neuroaffinity, for designing scaffolds in nerve tissue engineering.

Tissue-engineered bone grafts for osteoplasty in patients with cleft alveolus

Alveolar bone grafting is an integral part of the treatment concept in cleft palate patients. As an alternative to autogenous bone, tissue-engineered grafts have found some clinical application. The aim of the present study has been to compare ossification in the cleft area using tissue-engineered grafts in a case series of patients with ossification after transplantation of autogenous spongious bone as the gold standard in alveoloplasty. Eight children with complete cleft lips and cleft palates were included in the study. In four children (group A), the cleft defect was filled with tissue-engineered bone (autogenous osteoblasts cultured on demineralized bone matrix Osteovit(®)); as control in another 4 children (group B), the alveoloplasty was performed using spongious iliac bone. Preoperative and 6 months postoperative cone-beam computed tomography was performed, and volumes of the remaining cleft defects were calculated using 3D navigation software. Wound healing was uneventful in both groups. Six months postoperatively the mean volume of the cleft was 0.55±0.24cm(3) after grafting of tissue-engineered bone (group A) and 0.59±0.23cm(3) after transplantation of autogenous spongiosa. In group A, 40.9% of the cleft defect was ossified; in the control group (group B), 36.6%. Tissue-engineered bone is a promising alternative in alveolar bone grafting and no disadvantages were observed in comparison to the gold standard.

Effect of nanostructure on osteoinduction of porous biphasic calcium phosphate ceramics

In order to evaluate the effect of the nanostructure of calcium phosphate ceramics on osteoinductive potential, porous biphasic calcium phosphate (BCP) ceramics with a nano- or submicron structure were prepared via microwave sintering and compared to conventional BCP ceramics. The selective protein adsorption of bovine serum albumin and lysozyme (LSZ) and the osteogenic differentiation of human mesenchymal stem cells in vitro was investigated. Porous BCP nanoceramics showed higher ability to adsorb proteins, especially low molecular weight protein of LSZ, than conventional BCP ceramics, and the BCP nanoceramics promoted bone sialoprotein expression more than conventional BCP did. Further in vivo study to investigate ectopic bone formation and bone repair efficiency proved the highly osteoinductive potential of nanostructured BCP ceramics. The results suggest that nanostructured BCP ceramics have the potential to become a new generation of bioceramics for bone tissue engineering grafts.

Development of endothelium-denuded human umbilical veins as living scaffolds for tissue-engineered small-calibre vascular grafts

Tissue-engineered small-calibre vessel grafts may help to alleviate the lack of graft material for coronary and peripheral bypass grafting in an increasing number of patients. This study explored the use of endothelium-denuded human umbilical veins (HUVs) as scaffolds for vascular tissue engineering in a perfusion bioreactor. Vessel diameter (1.2 ± 0.4 mm), wall thickness (0.38 ± 0.09 mm), uniaxial ultimate failure stress (8029 ± 1714 kPa) and burst pressure (48.4 ± 20.2 kPa, range 28.4-83.9 kPa) were determined in native samples. The effects of endothelium removal from HUVs by enzymatic digestion, hypotonic lysis and dehydration were assessed. Dehydration did not significantly affect contractile function, tetrazolium dye reduction, mechanical strength and vessel structure, whereas the other methods failed in at least one of these parameters. Denudation by dehydration retained laminin, fibronectin, collagen and elastic fibres. Denuded HUVs were seeded in a perfusion bioreactor with either allogeneic HUVs endothelial cells or with saphenous vein endothelial cells harvested from patients with coronary artery disease. Seeding in a perfusion bioreactor resulted in a confluent monolayer of endothelial cells from both sources, as judged by histology and scanning electron microscopy. Seeded cells contained von Willebrand factor and CD31. In conclusion, denuded HUVs should be considered an alternative to decellularized blood vessels, as the process keeps the smooth muscle layer intact and functional, retains proteins relevant for biomechanic properties and for cell attachment and provides a suitable scaffold for seeding an autologous and flow-resistant endothelium.

Prefabrication of vascularized bone grafts using a combination of bone marrow mesenchymal stem cells and vascular bundles with β-tricalcium phosphate ceramics

Large bone defects are often treated with autologous vascularized bone grafts. These operations may be associated with donor site morbidities and a limited volume of harvested bone. To overcome such issues, we prefabricated vascularized bone grafts using a combination of bone marrow mesenchymal stem cells (BMSCs) and vascular bundles in a β-tricalcium phosphate ceramic (β-TCP). We used 15 New Zealand White rabbits as our experimental animals. Single photon-emission computed tomography and histologic analyses were used to evaluate angiogenesis and new bone formation of the bone grafts. The results showed that axial vessels not only promoted angiogenesis of the bone grafts, but also enhanced new bone formation. These findings suggested that the insertion of blood vessels into tissue-engineered bone grafts was an effective strategy for enhancing angiogenesis and bone formation and had potential significance for clinical applications.

Stem cells and vascular tissue-engineered grafts

Stem cells and vascular tissue-engineered grafts

Effect of Cyclic Strain on Cardiomyogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into cardiomyocyte-like lineage was investigated in this study. A stretching device was used to generate the cyclic strain for rBMSCs. Cardiomyogenic differentiation was evaluated using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western-blotting. The results demonstrated that appropriate cyclic strain treatment alone could induce cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, rBMSCs exposed to the strain stimulation expressed cardiomyocyte-related markers at a higher level than the shear stimulation. In addition, when rBMSCs were exposed to both strain and 5-azacytidine (5-aza), expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. These results suggest that cyclic strain is an important mechanical stimulus affecting the cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Myocardial Regeneration through Tissue Engineering

The overall goal of cardiac tissue engineering is to re‐establish the structure and function of infarcted myocardial tissue. Interdisciplinary research at the interface of stem cells and bioengineering holds promise that functional cardiac tissue grafts can be generated in vitro, and used for implantation as well as models to study cardiac development and disease. In all cases, the notion is that the biological potential of cardiovascular cells can be mobilized by providing controllable environments that regulate cell differentiation and assembly by factors present during native development. Key requirements for cardiac regeneration include an appropriate cardiac tissue matrix, electromechanical cell coupling, and functional vascularization. This talk will discuss some recent and ongoing work towards meeting these requirements. We will also discuss the important interactions between the engineered tissue grafts and the inflammatory environment of the infarct bed that largely determine the functional outcomes of tissue engineered heart repair.Funding: NIH (HL076485, EB002520, RR026244), Helmsley, NYSTEM

Bone Progenitors Produced by Direct Osteogenic Differentiation of the Unprocessed Bone Marrow Demonstrate High Osteogenic PotentialIn VitroandIn Vivo

Tissue-engineered bone grafts seeded with mesenchymal stem cells (MSCs) have been sought as a replacement for bone grafts currently used for bone repair. For production of osteogenic constructs, MSCs are isolated from bone marrow (BM) or other tissues, expanded in culture, then trypsinized, and seeded on a scaffold. Predifferentiation of seeded cells is often desired. We describe here bone progenitor cells (BPCs) obtained by direct osteogenic differentiation of unprocessed BM bypassing isolation of MSCs. Human BM aspirates were incubated for 2 weeks with a commonly used osteogenic medium (OM), except no fetal calf serum, serum substitutes, or growth factors were added, because responding stem and/or progenitor cells were present in the BM milieu. The adherent cells remaining after the culture medium and residual BM were washed out, expressed high levels of bone-specific alkaline phosphatase (ALP) on their surface, demonstrated high ALP activity, were capable of mineralization of the intercellular space, and expressed genes associated with osteogenesis. These parameters in BPCs were similar and even at higher levels compared to MSCs subjected to osteogenic differentiation for 2 weeks. The yield of BPCs per 1 mL BM was 0.71±0.39×106. In comparison, the yield of MSCs produced by adhesion of mononuclear cells derived from the same amount of BM and cultured in a commercial growth medium for 2 weeks was 0.3±0.17×106. When a scaffold was added to the BM-OM mixture, and the mixture was cultured in a simple rotational bioreactor; the resulting BPCs were obtained already seeded on the scaffold. BPCs seeded on scaffolds were capable of proliferation for at least 6 weeks, keeping high levels of ALP activity, expressing osteogenic genes, and mineralizing the scaffolds. Autologous rat BPCs seeded on various scaffolds were transplanted into critical-size calvarial defects. Six weeks after transplantation of polylactic acid/polyglycolic acid scaffolds, 76.1%±18.3% of the defects were filled with a new bone, compared to 37.9%±28.4% in the contralateral defects transplanted with the scaffolds without cells.

Recellularized nerve allografts with differentiated mesenchymal stem cells promote peripheral nerve regeneration

Chemical-extracted acellular nerve allografting, containing the natural nerve structure and elementary nerve extracellular matrix (ECM), has been used for peripheral nerve-defect treatment experimentally and clinically. However, functional outcome with acellular nerve allografting decreases with increased size of gap in nerve defects. Cell-based therapy is a good strategy for repairing long nerve defects. Bone-marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) can be induced to differentiate into cells with Schwann cell-like properties (BMSC–SCs or ADSC–SCs), which have myelin-forming ability in vitro and secrete trophic nerve growth factors. Here, we aimed to determine whether BMSC–SCs or ADSC–SCs are a promising cell type for enriching acellular grafts in nerve repair. We evaluated axonal regeneration distance by immunofluorescence staining after 2-week implantation. We used functional and histomorphometric analysis to evaluate 3-month regeneration of the novel cell-supplemented tissue-engineered nerve graft used to bridge a 15-mm-long sciatic nerve gap in rats. Introducing BMSC–SCs or ADSC–SCs to the acellular nerve graft promoted sciatic nerve regeneration and functional recovery. Nerve regeneration with BMSC–SCs or ADSC–SCs was comparable to that with autografting and Schwann cells alone and better than that with acellular nerve allografting alone. Differentiated bone-marrow-or adipose-derived MSCs may be a promising cell source for tissue-engineered nerve grafts and promote functional recovery after peripheral nerve injury.

Potential for polyhydroxyalkanoates and policaprolactone copolymer use as tissue-engineered scaffolds in cardiovascular surgery

The absence of reliably functioning small-diameter vascular grafts for coronary artery bypass graft surgery remains one of the most important issues of cardiovascular surgery. Tissue-engineered grafts have to be characterized by highly hemocompatible, biomechanical and biocompatible properties, be quickly biodegradable and have non-toxic degradation products. This article presents polyhydroxyalkanoate and policaprolactone main characteristics and evaluates their potential use as polymers for producing vascular grafts. Biocompatibility, good physical and mechanical properties of these polymers and their better performance in copolymer scaffolds were demonstrated.

Computer tomographic evaluation of talar edge configuration for osteochondral graft transplantation

To successfully surgically reconstruct osteochondral lesions of the talus, the exact three-dimensional (3D) configuration of the upper articular surface of the talus has to be respected. We assessed the talar geometry by measuring the coronal and sagittal talar edge radius and the frontal talar profile in multiplanar reconstructions of computer tomographic (CT) studies of 79 patients (83 feet) with a healthy ankle joint. An image visualization software designated for coordinate measurement was used to perform the measurement. In the coronal plane, the mean lateral talar edge radius was 4.0 mm and the medial 4.5 mm. In the sagittal planes the mean lateral talar edge radius was 20.3 mm, the radius of the sulcus 20.7 mm and the medial talar edge radius 20.4 mm. The talus showed a concave shape in coronal cuts. These results show a significant difference between medial and lateral talar edge configuration in coronal planes. The measurements of the lateral and medial sagittal radius and the mid-sagittal radius in the sulcus tali show no statistically significant difference. The depth of the talar sulcus shows no correlation to age or sex. Different sizes of custom-made tissue-engineered grafts according to the location of the osteochondral lesion at the talus are needed for exact surgical reconstruction of the anatomy. Osteochondral lesions are three dimensional; therefore, a 3D preoperative planning tool by CT scan or MRI is mandatory.

Optimization of Human Tendon Tissue Engineering

Tissue-engineered flexor tendon grafts may allow reconstruction of severe tendon losses. One critical factor is the optimization of cell proliferation and reseeding. Use of growth factors--basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and platelet-derived growth factor (PDGF)-BB--may improve culture conditions for human fibroblasts, tenocytes, and adipose-derived stem cells and increase repopulation of a tendon scaffold. All cell types were plated at a density of 10,000 cells per well and cultured in F12 media supplemented with varying concentrations of bFGF, IGF-1, and PDGF-BB. After 72 hours, cell proliferation was determined using the CellTiter assay. Human flexor tendon segments were acellularized and reseeded in a cell suspension of 5 × 10(5) cells/ml. After 5 days, tendon repopulation was determined using the MTS assay and histology. Statistical significance was determined with analysis of variance and a t test. For all cell types, there was enhanced proliferation with growth factors. Among single growth factors, PDGF-BB at 50 ng/ml was the most efficient stimulator of proliferation. With multiple growth factors, the optimal concentration was determined to be 5 ng/ml bFGF, 50 ng/ml IGF-1, and 50 ng/ml PDGF-BB (increase when compared with control: fibroblasts, 2.92-fold; tenocytes, 2.3-fold; and adipose-derived stem cells, 2.4-fold; p < 0.05). Tendons reseeded with this optimal combination of growth factors showed improved reseeding compared with the control group (fibroblasts, 2.01-fold; tenocytes, 1.78-fold; and adipose-derived stem cells, 1.76-fold; p < 0.05). bFGF, IGF-1, and PDGF-BB can be used to improve cellular proliferation and repopulation of an acellularized scaffold. The use of growth factors may be an important step in the tissue engineering of human flexor tendons.

V8 Presentation of a potential appropriate method for bulbar urethroplasty with tissue-engineered oral mucosa graft (MukoCell®)

V8 Presentation of a potential appropriate method for bulbar urethroplasty with tissue-engineered oral mucosa graft (MukoCell®)

Role of stem cells in the regeneration and repair of peripheral nerves

Peripheral nerve regeneration is a complex process, with Wallerian degeneration the most elementary reaction and Schwann cells playing an important role. In recent years, stem cells have been widely used to repair injured peripheral nerves. The sources of these stem cells are widespread and their effectiveness in the treatment of peripheral nerve injury may lie in their ability to differentiate into Schwann cells, secrete neurotrophic factors, and assist in myelin formation. Stem cells have been used as seed cells in tissue-engineered nerve grafts. The understanding of stem cell homing, novel repair material, and the ability to mobilize endogenous stem cells to assist peripheral nerve regeneration constitute a research direction of great interest.

418 細胞活性のための人工再生血管の最適構造設計手法の開発(OS-5 バイオメカニクス・バイオマテリアル(2))

418 細胞活性のための人工再生血管の最適構造設計手法の開発(OS-5 バイオメカニクス・バイオマテリアル(2))

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

Purpose 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. Materials and methods 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. Results 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. Conclusions 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.

Collagen‐immobilized patch for repairing small tympanic membrane perforations: In vitro and in vivo assays

Tympanic membrane (TM) perforation is still one of the most common otology complications. New designs of biomaterials, and lately tissue-engineered composites and grafts, have thoroughly revolutionized the management of TM perforation. In this study, we examined a biologically modified collagen-immobilized polydimethyl siloxane patch to repair TM perforation. In vitro potential of the aforementioned patch as a scaffold to support fibroblast cell growth and adhesion was assessed. An in vivo assay of the patch for initiating repair of TM perforations also was investigated. In vitro assay showed that the patch has significantly increased cell adhesion and growth in comparison with unmodified ones (p < 0.05). In vivo study also showed an overall closure rate of TM perforation of 70% and an average gain of 15.75 ± 4.29 dB in air-bone gap. This study shows that the preliminary in vivo evaluation of a modified siloxane patch in humans had promising results and is comparable to existing biomaterial patches.

The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers

Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic ( i.e., fibers collected randomly with no alignment) or anisotropic ( i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.