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1.
Injury ; 48(10): 2080-2088, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28842288

ABSTRACT

OBJECTIVES: Malignant neoplasms infiltrating the chest wall often requires resection of the thoracic wall. To replace the defect, Gore-Tex® is usually employed as the gold standard material, however, Gore-Tex® is inert and not degradable. Novel materials are nowadays available which allow a full bio-integration due to their non-toxic degradability. Additionally, stem cell seeding has the capacity to reduce inflammatory response towards such grafts, thus integrating it better into the host organism.


Subject(s)
Stem Cells/metabolism , Thoracic Wall/surgery , Tissue Scaffolds , Animals , Biocompatible Materials/pharmacology , Bone Regeneration , Disease Models, Animal , Male , Materials Testing , Mice , Mice, Inbred C57BL , Nanocomposites/chemistry , Plastic Surgery Procedures , Surgical Mesh , Tissue Engineering
2.
Macromol Biosci ; 16(7): 1048-63, 2016 07.
Article in English | MEDLINE | ID: mdl-27071839

ABSTRACT

Healing of tendon ruptures represents a major challenge in musculoskeletal injuries and combinations of biomaterials with biological factors are suggested as viable option for improved healing. The standard approach of repair by conventional suture leads to incomplete healing or rerupture. Here, a new elastic type of DegraPol® (DP), a polyester urethane, is explored as a delivery device for platelet-derived growth factor-BB (PDGF-BB) to promote tendon healing. Using emulsion electrospinning as an easy method for incorporation of biomolecules within polymers, DegraPol® supports loading and release of PDGF-BB. Morphological, mechanical and delivery device properties of the bioactive DP scaffolds, as well as differences arising due to different electrospinning parameters are studied. Emulsion electrospun DP scaffolds result in thinner fibers than pure DP scaffolds and experience decreased strain at break [%], but high enough for successful surgeon handling. PDGF-BB is released in a sustained manner from emulsion electrospun DP, but not completely, with still large amount of it being inside the polymeric fibers after 30 d. In vitro studies show that the bioactive scaffolds promote tenocyte proliferation in serum free and serum(+) conditions, demonstrating the potential of this surgeon-friendly bioactive delivery device to be used for tendon repair.


Subject(s)
Polyesters/administration & dosage , Polyurethanes/administration & dosage , Proto-Oncogene Proteins c-sis/administration & dosage , Rupture/drug therapy , Tendon Injuries/drug therapy , Becaplermin , Biocompatible Materials/administration & dosage , Biocompatible Materials/chemistry , Emulsions/administration & dosage , Emulsions/chemistry , Humans , Polyesters/chemistry , Proto-Oncogene Proteins c-sis/chemistry , Plastic Surgery Procedures , Rupture/physiopathology , Rupture/surgery , Tendon Injuries/physiopathology , Tendon Injuries/surgery , Tendons/physiopathology , Tendons/surgery , Wound Healing/drug effects
3.
J Mech Behav Biomed Mater ; 47: 124-134, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25916700

ABSTRACT

BACKGROUND: Perfusion bioreactors are used to solve problems in critical size bone tissue engineering. Biominerizable and biocompatible nanocomposites are suitable scaffold materials for this purpose because they offer mineral components in organic carriers. Human adipose derived stem cells (ASCs) can potentially be used to increase bone healing. MATERIALS AND METHODS: Electrospun nanocomposite disks of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) were seeded with ASCs and eight disks were stacked in a bioreactor running with normal culture. Under perfusion and uniaxial cyclic compression, load-displacement curves as a function of time were assessed. Stiffness and energy dissipation were recorded. Moreover, stem cell densities in the layers of the piled scaffold were determined as well as their morphologies and differentiation status. RESULTS: While the stiffness of the cell free constructs increased over time based on the transformation of the a-CaP nanoparticles into flake-like apatite, ASC-seeded constructs showed a constant stiffness. Stem cell density gradients had a linear increase from the bottom to the top of the pile (r(2)>0.95). Stem cells were getting more roundish at higher flow rates. Some osteogenesis was found upon osteopontin immunostaining, while no endothelial cell differentiation and no chondrogenesis was triggered. CONCLUSIONS: The fabrication of a critical size bone graft is presented based on a biominerizable bone-biomimetic nanocomposite with preserved stiffness when seeded with ASCs. The cell densities of ASCs inside the piled construct varied with a linear gradient. Beginning osteogenesis was triggered by the dynamic culture conditions including perfusion and compression.


Subject(s)
Biomimetic Materials/pharmacology , Bioreactors , Minerals/metabolism , Nanocomposites , Perfusion , Stem Cells/cytology , Stem Cells/drug effects , Adipose Tissue/cytology , Biomimetic Materials/chemistry , Calcium Phosphates/chemistry , Cell Count , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Humans , Lactic Acid/chemistry , Nanoparticles/chemistry , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Stem Cells/metabolism , Tissue Engineering , Tissue Scaffolds/chemistry , Weight-Bearing
4.
Injury ; 45(6): 974-80, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24650943

ABSTRACT

BACKGROUND: Fractures with a critical size bone defect are associated with high rates of delayed- and non-union. The treatment of such complications remains a serious issue in orthopaedic surgery. Adipose derived stem cells (ASCs) combined with biomimetic materials can potentially be used to increase fracture healing. Nevertheless, a number of requirements have to be fulfilled; in particular, the insufficient vascularisation of the bone constructs. Here, the objectives were to study the impact of ASC-derived osteoblasts on ASC-derived endothelial cells in a 3D co-culture and the effect of 40wt% of amorphous calcium phosphate nanoparticles on the proliferation and differentiation of ASC-derived endothelial cells when present in PLGA. MATERIALS AND METHODS: Five primary ASC lines were differentiated towards osteoblasts (OBs) and endothelial cells (ECs) and two of them were chosen based on quantitative PCR results. Either a mono-culture of ASC-derived EC or a co-culture of ASC-derived EC with ASC-derived OB (1:1) was seeded on an electrospun nanocomposite of poly-(lactic-co-glycolic acid) and amorphous calcium phosphate nanoparticles (PLGA/a-CaP; reference: PLGA). The proliferation behaviour was determined histomorphometrically in different zones and the expression of von Willebrand Factor (vWF) was quantified. RESULTS: Independently of the fat source (biologic variability), ASC-derived osteoblasts decelerated the proliferation behaviour of ASC-derived endothelial cells in the co-culture compared to the mono-culture. However, expression of vWF was clearly stronger in the co-culture, indicating further differentiation of the ASC-derived EC into the EC lineage. Moreover, the presence of a-CaP nanoparticles in the scaffold slowed the proliferation behaviour of the co-culture cells, too, going along with a further differentiation of the ASC-derived OB, when compared to pure PLGA scaffolds. CONCLUSIONS: This study revealed significant findings for bone tissue-engineering. Co-cultures of ASC-derived EC and ASC-derived OB stimulate each other's further differentiation. A nanocomposite with a-CaP nanoparticles offers higher mechanical stability, bioactivity and osteoconductivity compared to mere PLGA and can easily be seeded with pre-differentiated EC and OB.


Subject(s)
Adipose Tissue/cytology , Biomimetic Materials , Endothelial Cells/cytology , Fractures, Bone/pathology , Fractures, Ununited/pathology , Osteoblasts/cytology , Tissue Engineering/methods , Biomechanical Phenomena , Calcium Phosphates/metabolism , Cell Differentiation , Cell Line , Cell Proliferation , Cells, Cultured , Coculture Techniques , Endothelial Cells/metabolism , Humans , Nanocomposites , Osteoblasts/metabolism , Osteogenesis , Stem Cells , Tissue Scaffolds
5.
Cytotherapy ; 15(9): 1098-105, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23800730

ABSTRACT

BACKGROUND AIMS: Adipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed. METHODS: The surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined. RESULTS: Both adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site. CONCLUSIONS: The floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate.


Subject(s)
Adipose Tissue/physiology , Stem Cells/physiology , Adipose Tissue/metabolism , Adult , Aged , Antigens, CD/metabolism , Body Mass Index , Cell Culture Techniques/methods , Cell Proliferation , Cells, Cultured , Female , Humans , Male , Middle Aged , Stem Cells/metabolism
6.
Injury ; 43(10): 1689-97, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22769980

ABSTRACT

For tissue engineering of critical size bone grafts, nanocomposites are getting more and more attractive due to their controllable physical and biological properties. We report in vitro and in vivo behaviour of an electrospun nanocomposite based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) seeded with human adipose-derived stem cells (ASC) compared to PLGA. Major findings were that cell attachment, three-dimensional ingrowth and proliferation were very good on both materials. Cell morphology changed from a spindle-shaped fibroblast-like form to a more roundish type when ASC were seeded on PLGA, while they retained their morphology on PLGA/a-CaP. Moreover, we found ASC differentiation to a phenotype committed towards osteogenesis when a-CaP nanoparticles were suspended in normal culture medium without any osteogenic supplements, which renders a-CaP nanoparticles an interesting osteoinductive component for the synthesis of other nanocomposites than PLGA/a-CaP. Finally, electrospun PLGA/a-CaP scaffold architecture is suitable for a rapid and homogenous vascularisation confirmed by a complete penetration by avian vessels from the chick chorioallantoic membrane (CAM) within one week.


Subject(s)
Adipose Tissue/cytology , Bone Transplantation/methods , Calcium Phosphates/chemistry , Lactic Acid/chemistry , Nanocomposites/chemistry , Osteoblasts , Osteogenesis , Polyglycolic Acid/chemistry , Stem Cell Transplantation , Tissue Engineering , Tissue Scaffolds , Biocompatible Materials , Cell Proliferation , Female , Humans , Male , Nanoparticles , Osteogenesis/physiology , Polylactic Acid-Polyglycolic Acid Copolymer
7.
Tissue Eng Part A ; 17(3-4): 291-9, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20799888

ABSTRACT

Tissue engineering of bone grafts was addressed in a critical-sized model on the chick chorioallantoic membrane model, using DegraPol(®) foam as scaffold material. The scaffolds were seeded with cultures of human osteoblasts and human endothelial cells, respectively, or with a co-culture of the two cell types (control: no cells). In vitro samples (7 days cultivation) and ex vivo chorioallantoic membrane model samples at incubation day 15 were analyzed by high-field magnetic resonance imaging (MRI) and histology. The co-culture system performed best with respect to perfusion, as assessed by contrast-enhanced MRI using gadolinium-diethylene-triamine-pentaacetic acid (DTPA). The scaffold seeded by the co-culture supported an increased vascular ingrowth, which was confirmed by histological analysis. DegraPol foam is a suitable scaffold for bone tissue engineering and the MRI technique allows for nondestructive and quantitative assessment of perfusion capability during early stages of bone forming constructs.


Subject(s)
Bone Transplantation/instrumentation , Bone and Bones/blood supply , Capillaries/growth & development , Endothelial Cells/cytology , Osteoblasts/cytology , Tissue Scaffolds , Adolescent , Biocompatible Materials/chemistry , Bone Development/physiology , Bone and Bones/cytology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Coculture Techniques/instrumentation , Endothelial Cells/physiology , Female , Gases/chemistry , Humans , Magnetic Resonance Imaging , Osteoblasts/physiology
8.
Tissue Eng Part A ; 15(9): 2471-80, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19292679

ABSTRACT

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.


Subject(s)
Fluorocarbons/pharmacology , Oxygen/metabolism , Tissue Engineering , Trachea/drug effects , Trachea/physiology , Animals , Chondrocytes/cytology , Chondrocytes/drug effects , Chorioallantoic Membrane/cytology , Chorioallantoic Membrane/drug effects , Chorioallantoic Membrane/transplantation , Dermis/drug effects , Epithelium/drug effects , Foreign-Body Reaction/pathology , Glycosaminoglycans/metabolism , Humans , Hydrocarbons, Brominated , Microdialysis , Partial Pressure , Polyesters/pharmacology , Polyurethanes/pharmacology , Rats , Sus scrofa , Tissue Scaffolds
9.
Tissue Eng ; 13(10): 2483-93, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17655485

ABSTRACT

The advantage of using anatomically shaped scaffolds as compared to modeled designs was investigated and assessed in terms of cartilage formation in an artificial tracheal construct. Scaffolds were rapid prototyped with a technique named three-dimensional fiber deposition (3DF). Anatomical scaffolds were fabricated from a patient-derived computerized tomography dataset, and compared to cylindrical and toroidal tubular scaffolds. Lewis rat tracheal chondrocytes were seeded on 3DF scaffolds and cultured for 21 days. The 3-(4,5-dimethylthiazol-2yl)-2,5-dyphenyltetrazolium bromide (MTT) and sulfated glycosaminoglycan (GAG) assays were performed to measure the relative number of cells and the extracellular matrix (ECM) formed. After 3 weeks of culture, the anatomical scaffolds revealed a significant increase in ECM synthesis and a higher degree of differentiation as shown by the GAG/MTT ratio and by scanning electron microscopy analysis. Interestingly, a lower scaffold's pore volume and porosity resulted in more tissue formation and a better cell differentiation, as evidenced by GAG and GAG/MTT values. Scaffolds were compliant and did not show any signs of luminal obstruction in vitro. These results may promote anatomical scaffolds as functional matrices for tissue regeneration not only to help regain the original shape, but also for their improved capacity to support larger tissue formation.


Subject(s)
Artificial Organs , Cell Culture Techniques/methods , Chondrocytes/cytology , Chondrocytes/physiology , Polyesters/chemistry , Polyethylene Glycols/chemistry , Trachea/cytology , Trachea/growth & development , Animals , Cells, Cultured , Rats , Rats, Inbred Lew , Tissue Engineering
10.
Eur J Cardiothorac Surg ; 31(5): 806-11, 2007 May.
Article in English | MEDLINE | ID: mdl-17320405

ABSTRACT

OBJECTIVE: To test the effects of a continuous medium flow inside DegraPol scaffolds on the reepithelialization and revascularization processes of a tissue-engineered trachea prosthesis. METHODS: In this proof-of-principle study a continuous medium flow was maintained within a tubular DegraPol scaffold by an inserted porous catheter connected to a pump system. The impact of the intra-scaffold medium flow on the survival of a tracheal epithelial sheet wrapped around and on chondrocyte delivery to the DegraPol scaffold was studied. In the chick embryo, chorioallantoic membrane (CAM) model angiogenesis within the biomaterial was investigated. RESULTS: Scanning electronic microscopy (SEM) images showed an intact epithelial layer after a 2-week support by continuous medium flow underneath. On histology, three-dimensional cell growth was detected in the continuous delivery group. The CAM assay showed that angiogenesis was enhanced within the DegraPol scaffolds when vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) was added to the perfusate. CONCLUSIONS: Taken together, these results demonstrated that the built-in perfusion system within DegraPol scaffolds was able to maintain an intact tracheal epithelial layer, allowed a continuous delivery of cells, and kept an efficient VEGF/VPF expression level which accelerated angiogenic response in the CAM assay. This design combines the in vitro and in vivo parts of tissue engineering and offers the possibility to be used as an in vivo bioreactor implanted for the tissue-engineered reconstruction of trachea and of other organs.


Subject(s)
Tissue Engineering/methods , Trachea/blood supply , Vascular Endothelial Growth Factor A/administration & dosage , Animals , Bioprosthesis , Bioreactors , Cell Count , Cell Line , Chondrocytes/physiology , Epithelial Cells/physiology , Epithelial Cells/ultrastructure , Humans , Microscopy, Electron, Scanning/methods , Models, Biological , Neovascularization, Physiologic/physiology , Pilot Projects , Polyesters , Polyurethanes , Rats , Trachea/ultrastructure
11.
Eur J Cardiothorac Surg ; 24(2): 201-7, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12895608

ABSTRACT

OBJECTIVE: To date numerous attempts have been undertaken to conquer the challenging problem of reconstructing long segmental tracheal defects, as yet without lasting success. Recently, employing concepts of tissue engineering in animals, cartilage-like constructs were transplanted in vivo. However, both the feasibility of fabricating tracheal replacements and the use of human tracheal chondrocytes (HTC) for tissue engineering are still under investigation. In this study, we optimized isolation and cultivation techniques for human tracheal cartilage, assessing the feasibility of seeding these cells onto a novel, three-dimensional (3-D) polyester-urethane polymer (DegraPol). METHODS: Human tracheal cartilage was harvested from the trachea of lung donors, digested in 0.3% collagenase II, and the condrocytes serially passaged every 7-9 days. Cells were also cultivated over agar plate during the total 6-8 weeks expansion phase. Thereafter, chondrocytes were seeded onto DegraPol (pore sizes 150-200 microm) with a seeding density of 2.4 x 10(7)/ml, and chondrocyte-polymer constructs maintained during in vitro static culture. RESULTS: HTC displayed stable proliferation kinetics in monolayer culture with positive expression of collagen type II. Following polymer seeding, both cellular proliferation and extracellular matrix (ECM) production, as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and glycosaminoglycan assays, continued over extended culture. Active growth of HTC on DegraPol was further demonstrated by Alcian blue staining, with the histomorphological appearance of the construct resembling that of native cartilage. Scanning electron microscopy showed chondrocyte growth and ECM synthesis both on the surface and inside the porous scaffold, with a dense cell layer on the surface of the scaffold and a lower cell distribution in the scaffold's interior. CONCLUSIONS: The harvested chondrocytes from human trachea cartilage expand well in vitro and possess the ability to form new cartilage-like tissue when seeded onto DegraPol matrix. However, improved culture conditions are needed to permit cellular growth throughout cell-polymer constructs.


Subject(s)
Chondrocytes , Polyesters , Polyurethanes , Tissue Engineering/methods , Trachea , Absorbable Implants , Adult , Biomarkers/analysis , Cell Culture Techniques , Cell Division , Chondrocytes/ultrastructure , Collagen Type II/analysis , Feasibility Studies , Humans , Microscopy, Electron, Scanning
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