Tissue Engineering Purposes Research Articles

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Impact of Detergent-Based Decellularization Methods on Porcine Tissues for Heart Valve Engineering

To date an optimal decellularization protocol of heart valve leaflets (HVL) and pericardia (PER) with an adequate preservation of the extracellular matrix (ECM) is still lacking. This study compares a 4 day Triton X-100-based protocol with faster SDC-based protocols for the decellularization of cardiac tissues. Decellularized and non-treated HVL and PER were processed for histological, biochemical and mechanical analysis to determine the effect of these agents on the structure, ECM components, and biomechanical properties. Tissues treated with SDC-based protocols still showed nuclear material, whereas tissues treated with Triton X-100 1%+ENZ±TRYP were completely cell free. For both decellularized tissues, an almost complete washout of glycosaminoglycans, a reduction of soluble collagen and an alteration of the surface ultrastructure was observed. Interestingly, only the elastic fibers of pericardial tissue were affected and this tissue had a decreased maximum load. This study showed that both detergents had a similar impact on the ECM. However, Triton X-100 1% +DNase/RNase (ENZ)±Trypsin (TRYP) is the only protocol that generated completely cell free bioscaffolds. Also, our study clearly demonstrated that the decellularization agents have more impact on pericardial tissues than on heart valve leaflets. Thus, for the purpose of tissue engineering of heart valves, it is advisable to use valvular rather than pericardial matrices.

Application of chitin-based biomaterials to regulate the basement membrane dynamics for engineering epithelial structure formation

Event Abstract Back to Event Application of chitin-based biomaterials to regulate the basement membrane dynamics for engineering epithelial structure formation Tsung-Lin Yang1* and Ya-Chuan Hsiao2* 1 National Taiwan University, Otolaryngology, Taiwan 2 Zhongxing Branch, Taipei City Hospital, Department of Ophthalmology, Taiwan Tissue structure is important for inherent physiological function of epithelial organs, and also critically required in organ regeneration when tissue is engineered. Many essential organs responsible for secretion, nutrition supply, or metabolite exchange, are featured and benefited by ramified tissue architecture during organogenesis. For the purpose of tissue engineering, formation of tissue structures are still challenging. The salivary gland is a typical epithelial organ important for saliva secretion and regulation. The salivary glands develop from epithelial-mesenchymal interaction, and accordingly depend on the support of basement membrane (BM). It is therefore hypothesized that regulation of BM components by biomaterial may facilitate the tissue structure formation. Chitin-based biomaterials had been demonstrated to be competent in facilitating tissue structural formation of the salivary glands. Using the developing submandibular gland as a model, it was found that chitosan effect diminished when BM components were removed from cultured SMG explants. With chitosan, BM components and receptors increased, and expressed in tissue-specific manners beneficial for SMG branching. Chitosan effect decreased when either BM components or receptors were removed, and reduced as well when downstream signaling of BM components and receptors was blocked. Our results revealed that the morphogenetic effect of chitosan on salivary glands branching is through the regulation of the dynamics of BM components. This study revealed the underlying mechanism accounting for chitin-based biomaterial effect in engineering branching structure formation of the salivary glands, which paves ways for further optimization and application of chitin-based biomaterials in facilitating structure formation of epithelial organs. Keywords: Tissue Engineering, Mechanism, biomaterial, complex tissue orgnization Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Regenerative medicine: biomaterials for control of tissue induction Citation: Yang T and Hsiao Y (2016). Application of chitin-based biomaterials to regulate the basement membrane dynamics for engineering epithelial structure formation. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01216 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Tsung-Lin Yang, National Taiwan University, Otolaryngology, Taipei, Taiwan, Email1 Dr. Ya-Chuan Hsiao, Zhongxing Branch, Taipei City Hospital, Department of Ophthalmology, Taipei, Taiwan, Email2 Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tsung-Lin Yang Ya-Chuan Hsiao Google Tsung-Lin Yang Ya-Chuan Hsiao Google Scholar Tsung-Lin Yang Ya-Chuan Hsiao PubMed Tsung-Lin Yang Ya-Chuan Hsiao Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Preliminary study of surface modification of 3D Poly (ɛ - caprolactone) scaffolds by ultrashort laser irradiation

Three - dimensional poly (e- caprolactone) (PCL) scaffolds as suitable biocompatible material for manufacturing tissue replacements are utilized for tissue engineering purposes. The porous structures are fabricated by rapid prototyping method (Bioscaffolder) based on hypodermic dispensing process. The consecution of experiments demonstrated the possibility on creation of surface micro formations, applying different laser fluences, at 1 kHz repetition rate for fixed time of exposure 1 sec at 800 nm central wavelength. The combination of both methods offers possibilities for successful production of 3D matrices with modified surfaces. The obtained results of laser - induced surface modifications of PCL demonstrate the potential of the method to microprocess this kind of material for possible applications in regenerative medicine.

Magnetic Bacterial Nanocellulose for neurovascular reconstruction in cerebral aneurysm treatment applications

Event Abstract Back to Event Magnetic Bacterial Nanocellulose for neurovascular reconstruction in cerebral aneurysm treatment applications Sandra L Arias1, 2*, Akshath Shetty2, 3, Angana Senpan2, Monica Echeverry-Rendon4 and Jean Paul Allain2, 3 1 University of Illinois at Urbana-Champaign, Department of Bioengineering, United States 2 University of Illinois at Urbana-Champaign, Micro and Nanotechnology Laboratory, United States 3 University of Illinois at Urbana-Champaign, Department of Nuclear, Plasma and Radiological Engineering, United States 4 University of Antioquia, Department of Materials Engineering, Colombia Introduction: Cerebral aneurysms are abnormal blood pockets that form outside of the arterial wall in the brain. The main obstacle in aneurysmal neuroendovascular reconstruction approaches is the delayed closure of the aneurysm due to poor cell retention[1]. We hypothesized that a magnetic bacterial nanocellulose (MBNC) can improve local cell retention at the site of the aneurysmal neck defect via focal magnetic attractive forces. Bacterial nanocellulose (BNC) is a natural hydrogel produced by A. Xylinum, which has high mechanical properties (114 GPa for a single filament of BNC)[2], and is biocompatible[3]. To test the feasibility of our approach, we precipitated iron oxide nanoparticles (IONPs) in BNC pellicles to yield a magnetic hydrogel, and characterized the resulting material via XPS, SEM and vibrating sample magnetometer (VSM). We additionally introduced cell adhesion sites (collagen) on the MBNC via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) activation of the cellulose fibrils. A cytotoxicity test based on intracellular esterase activity was used to test the biocompatibility of the MBNC pellicles. Materials and Methods: Magnetic functionalization: The MBNC was prepared by in-situ precipitation of Fe2+ and Fe3+ ferrous ions in never-dried BNC[4]. Collagen bio-conjugation: 100 ul of CDAP solution was added on top of BNC and MBNC pellicles and allow to react for 1 min. Subsequently, 0.1 N of HCl was added per sample to stop the activation. The pellicles were washed with PBS and 200 ul of collagen was added on top per sample. The biocompatibility of the samples was tested via calcein/ethidium homodiamer-1 to mark healthy and apoptotic human smooth muscle cells (HSMCs) respectively. MBNC characterization: XPS was employed to reveal the chemical composition both of MBNC and collagen-bioconjugated MBNC pellicles. Magnetic strength via VSM was used to measure the magnetic strength of the MBNC. SEM was used to examine the morphology of the MBNC. Results and Discussion: Fig. 1A shows the set-up used in the fabrication of MBNC. The BNC acquires a black color after incorporation of IONPs, which are dispersed along the BNC fibers or forming aggregates (Fig. 1B-D). Compared with the XPS of BNC (Fig. 2), the spectrum of MBNC shows a significant new signal at a binding energy (BE) of 722 and 709 eV, which is the characteristic peak of Fe 2p1/2 and Fe 2p3/2. The XPS on collagen-conjugated MBNC resulted on the detection of nitrogen, which can be interpreted as an indirect measure of the protein content. Fig. 3 shows the esterase activity measured via calcein (green) of HSMCs culture on MBNC for 24 hours. Both samples show a high HSMC viability and confluence, indicanting that the MBNC and collagen-bioconjugated MBNC did not have negative detrimental effects on HSMCs under these experimental conditions. . The VSM plot of MBNC provide evidence that IONPS are superparamagnetic at room temperature. Conclusion: Our results show that we successfully incorporated Fe3O4 nanoparticles and cell-adhesion sites on the BNC. The VSM data showed that MBNC had a magnetic saturation of (0.054), which is above of the value considered to have satisfactory magnetic strength for tissue engineering purposes (0.049 emu/g). This work was funded by Department of Defense under contract No. W81XWH-11-2-0067

Amorphous polyphosphate–hydroxyapatite: A morphogenetically active substrate for bone-related SaOS-2 cells in vitro

There is increasing evidence that inorganic calcium-polyphosphates (polyP) are involved in human bone hydroxyapatite (HA) formation. Here we investigated the morphology of the particles, containing calcium phosphate (CaP) with different concentrations of various Na–polyP concentrations, as well as their effects in cell culture. We used both SaOS-2 cells and human mesenchymal stem cells. The polymeric phosphate readily binds calcium ions under formation of insoluble precipitates. We found that addition of low concentrations of polyP (<10wt.%, referred to the CaP deposits) results in an increased size of the HA crystals. Surprisingly, at higher polyP concentrations (>10wt.%) the formation of crystalline HA is prevented and amorphous polyP/HA hybrid particles with a size of ≈50nm are formed, most likely consisting of polyP molecules linked via Ca2+ bridges to the surface of the CaP deposits. Further studies revealed that the polyP–CaP particles cause a strong upregulation of the expression of the genes encoding for two marker proteins of bone formation, collagen type I and alkaline phosphatase. Based on their morphogenetic activity the amorphous polyP–CaP particles offer a promising material for the development of bone implants, formed from physiological inorganic precursors/polymers. Statement of significanceHydroxyapatite (HA) is a naturally occurring mineral of vertebrate bone. Natural HA, a bio-ceramic material which is crystalline to different scale, has been used as a biomaterial to fabricate scaffolds for in situ bone regeneration and other tissue engineering purposes. In contrast to natural HA, synthetic apatite is much less effective. In general, while HA is bioactive, its interaction and biocompatibility with existing bone tissue is low. These properties have been attributed to a minimal degradability in the physiological environment. In the present study we introduce a new Ca–phosphate (CaP) fabrication technology, starting from calcium chloride and dibasic ammonium phosphate with the HA characteristic Ca/P molar ratio of 10:6 and report that after addition >10% (by weight) of polyphosphate (polyP) amorphous CaP/HA samples were obtained. Those samples elicits strong morphogenetic activity let us to conclude that polyP/HA-based material might be beneficial for application as bone substitute implant.

In vitro and in vivo bone formation potential of surface calcium phosphate-coated polycaprolactone and polycaprolactone/bioactive glass composite scaffolds

In this study, polycaprolactone (PCL)-based composite scaffolds containing 50wt% of 45S5 Bioglass® (45S5) or strontium-substituted bioactive glass (SrBG) particles were fabricated into scaffolds using an additive manufacturing technique for bone tissue engineering purposes. The PCL scaffolds were surface coated with calcium phosphate (CaP) to enable further comparison of the osteoinductive potential of different scaffolds: PCL (control), PCL/CaP-coated, PCL/50-45S5 and PCL/50-SrBG scaffolds. The PCL/50-45S5 and PCL/50-SrBG composite scaffolds were reproducibly manufactured with a morphology highly resembling that of PCL only scaffolds. However, 50wt% loading of the bioactive glass (BG) particles into the PCL bulk decreased the scaffold’s compressive Young’s modulus. Coating of PCL scaffolds with CaP had a negligible effect on the scaffold’s porosity and compressive Young’s modulus. When immersed in culture media, BG dissolution ions (Si and Sr) were detected for up to 10weeks in the immersion media and surface precipitates were formed on both PCL/50-45S5 and PCL/50-SrBG scaffolds’ surfaces, indicating good in vitro bioactivity. In vitro cell studies were conducted using sheep bone marrow stromal cells (BMSCs) under non-osteogenic or osteogenic conditioned media, and under static or dynamic culture environments. All scaffolds were able to support cell adhesion, growth and proliferation. However, when cultured in non-osteogenic media, only PCL/CaP, PCL/50-45S5 and PCL/50-SrBG scaffolds showed an up-regulation of osteogenic gene expression. Additionally, under a dynamic culture environment, the rate of cell growth, proliferation and osteoblast-related gene expression was enhanced across all scaffold groups. Subsequently, PCL/CaP, PCL/50-45S5 and PCL/50-SrBG scaffolds, with or without seeded cells, were implanted subcutaneously into nude rats for the evaluation of osteoinductivity potential. After 8 and 16weeks, host tissue infiltrated well into the scaffolds, but no mature bone formation was observed in any scaffolds groups. Statement of significanceThis novelty of this research work is that it provide a comprehensive comparison, both in vitro and in vivo, between 3 different composite materials widely used in the field of bone tissue engineering for their bone regeneration capabilities. The materials used in this study include polycaprolactone, 45S5 Bioglass, strontium-substituted bioactive glass and calcium phosphate. Additionally, the composite materials were fabricated into the form of 3D scaffolds using additive manufacturing technique, a widely used technique in tissue engineering.

Formation of size-controllable spheroids using gingiva-derived stem cells and concave microwells: Morphology and viability tests.

Human mesenchymal stem cells have previously been isolated and characterized from the gingiva, and gingiva-derived stem cells have been applied for tissue engineering purposes. The present study was performed to generate size-controllable stem cell spheroids using concave microwells. Gingiva-derived stem cells were isolated, and the stem cells of 1×105 (group A) or 2×105 (group B) cells were seeded in polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres was viewed under an inverted microscope, and the changes in the diameter and cell viability were analyzed. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A compared to group B. No significant changes in shape or diameter were noted with increases in incubation time. Cell viability was higher in group B at each time point when compared with group A. Within the limits of the study, the size-controllable stem cell spheroids could be generated from gingival cells using microwells. The shape of the spheroids and their viability were clearly maintained during the experimental periods.

Fabrication of Poly-l-lactic Acid/Dicalcium Phosphate Dihydrate Composite Scaffolds with High Mechanical Strength-Implications for Bone Tissue Engineering.

Scaffolds were fabricated from poly-l-lactic acid (PLLA)/dicalcium phosphate dihydrate (DCPD) composite by indirect casting. Sodium citrate and PLLA were used to improve the mechanical properties of the DCPD scaffolds. The resulting PLLA/DCPD composite scaffold had increased diametral tensile strength and fracture energy when compared to DCPD only scaffolds (1.05 vs. 2.70 MPa and 2.53 vs. 12.67 N-mm, respectively). Sodium citrate alone accelerated the degradation rate by 1.5 times independent of PLLA. Cytocompatibility of all samples were evaluated using proliferation and differentiation parameters of dog-bone marrow stromal cells (dog-BMSCs). The results showed that viable dog-BMSCs attached well on both DCPD and PLLA/DCPD composite surfaces. In both DCPD and PLLA/DCPD conditioned medium, dog-BMSCs proliferated well and expressed alkaline phosphatase (ALP) activity indicating cell differentiation. These findings indicate that incorporating both sodium citrate and PLLA could effectively improve mechanical strength and biocompatibility without increasing the degradation time of calcium phosphate cement scaffolds for bone tissue engineering purposes.

Matrix Addressing of an Electronic Surface Switch Based on a Conjugated Polyelectrolyte for Cell Sorting

Spatial control of cell detachment is potentially of great interest when selecting cells for clonal expansion and in order to obtain a homogeneous starting population of cells aimed for tissue engineering purposes. Here, selective detachment and cell sorting of human primary keratinocytes and fibroblasts is achieved using thin films of a conjugated polymer. Upon electrochemical oxidation, the polymer film swells, cracks, and finally detaches taking cells cultured on top along with it. The polymer can be patterned using standard photolithography to fabricate a cross‐point matrix with polymer pixels that can be individually addressed and thus detached. Detachment occurs above a well‐defined threshold of +0.7 V versus Ag/AgCl, allowing the use of a relatively simple and easily manufactured passive matrix‐addressing configuration, based on a resistor network, to control the cell‐sorting device.

Comparative evaluation of invivo biocompatibility and biodegradability of regenerated silk scaffolds reinforced with/without natural silk fibers.

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving invitro and invivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration. spjba;30/6/793/FIG10885328215601925F1fig1-0885328215601925Figure 1.Illustrative summary of the main methods and findings.RS: regenerated silk; RSF: regenerated fibroin/ silk fiber composite scaffolds; H&E: Hematoxylin and eosin; COX-1: Cyclooxygenase.

Uniform and electrically conductive biopolymer-doped polypyrrole coating for fibrous PLA.

Three-dimensional, fibrous scaffolds can be easily fabricated from polylactide (PLA) using melt spinning and textile techniques. However, the surface properties of PLA scaffolds are not ideal for tissue engineering purposes. Furthermore, electrically conducting scaffolds are required to deliver electrical stimulation to cells. In this study, uniform, electrically conducting polypyrrole (PPy) coatings were fabricated on biodegradable PLA fibers. Biopolymer dopants-hyaluronic acid (HA) and chondroitin sulfate (CS)-were compared, and a PPy/CS composition was analyzed further. The effect of the oxidative polymerization conditions on the PLA fibers and CS counterion was studied. Furthermore, the initial molecular weight of CS and its degree of polymerization were determined. Our experiments showed that the molecular weight of CS decreases under oxidizing conditions but that the decay is not significant with the short polymerization process we used. The coating process was transferred to nonwoven PLA fabrics, and the stability of PPy/CS coating was studied during in vitro incubation in phosphate buffer solution at physiological temperature. The conductivity and surface roughness of the coating decayed during the 20-day incubation. The mechanical strength, however, remained at the initial level. Thus, the fabricated structures are suitable for short-term electrical stimulation adequate to promote cell functions in specific cases. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1721-1729, 2016.

Dental Pulp Cell Behavior in Biomimetic Environments

There is emerging recognition of the importance of a physiologically relevant in vitro cell culture environment to promote maintenance of stem cells for tissue engineering and regenerative medicine purposes. In vivo, appropriate cellular cues are provided by local tissue extracellular matrix (ECM), and these are not currently recapitulated well in vitro using traditional cultureware. We therefore hypothesized that better replication of the in vivo environment for cell culture and differentiation could be achieved by culturing dental pulp cells with their associated ECM. Primary dental pulp cells were subsequently seeded onto pulp-derived ECM-coated cultureware. While at up to 24 h they exhibited the same level of adherence as those cells seeded on tissue culture–treated surfaces, by 4 d cell numbers and proliferation rates were significantly decreased in cells grown on pulp ECM compared with controls. Analysis of stem cell and differentiation marker transcripts, as well as Oct 3/4 protein distribution, supported the hypothesis that cells cultured on ECM better maintained a stem cell phenotype compared with those cultured on standard tissue culture–treated surfaces. Subsequent differentiation analysis of cells cultured on ECM demonstrated that they exhibited enhanced mineralization, as determined by alizarin red staining and mineralized marker expression. Supplementation of a 3% alginate hydrogel with pulp ECM components and dental pulp cells followed by differentiation induction in mineralization medium resulted in a time-dependent mineral deposition at the periphery of the construct, as demonstrated histologically and using micro–computed tomography analysis, which was reminiscent of tooth structure. In conclusion, data indicate that culture of pulp cells in the presence of ECM better replicates the in vivo environment, maintaining a stem cell phenotype suitable for downstream tissue engineering applications.

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with l-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.

Inhibition of differentiation and function of osteoclasts by dimethyl sulfoxide (DMSO).

Dimethyl sulfoxide (DMSO) is an FDA-approved organosulfur solvent that is reported to have therapeutic value in osteoarthritis and osteopenia. DMSO is used as a cryoprotectant for the cryopreservation of bone grafts and mesenchymal stem cells which are later used for bone repair. It is also used as a solvent in the preparation of various scaffolds used for bone tissue engineering purposes. DMSO has been reported to inhibit osteoclast formation in vitro but the mechanism involved has remained elusive. We investigated the effect of DMSO on osteoclast differentiation and function using a conventional model system of RAW 264.7 cells. The differentiation of RAW 264.7 cells was induced by adding 50 ng/ml RANKL and the effect of DMSO (0.01 and 1% v/v) on RANKL-induced osteoclastogenesis was investigated. Addition of 1% DMSO significantly inhibited RANKL-induced formation of TRAP+, multinucleated, mature osteoclasts and osteoclast late-stage precursors (c-Kit(-) c-Fms(+) Mac-1(+) RANK(+)). While DMSO did not inhibit proliferation per se, it did inhibit the effect of RANKL on proliferation of RAW 264.7 cells. Key genes related to osteoclast function (TRAP, Integrin αVβ3, Cathepsin K and MMP9) were significantly down-regulated by DMSO. RANKL-induced expression of RANK gene was significantly reduced in the presence of DMSO. Our data, and reports from other investigators, that DMSO enhances osteoblastic differentiation of mesenchymal stem cells and also prevents bone loss in ovarietcomized rats, suggest that DMSO has tremendous potential in the treatment of osteoporosis and bone diseases arising from uncontrolled activities of the osteoclasts.

Cytocompatibility of a conductive nanofibrous carbon nanotube/poly (L-Lactic acid) composite scaffold intended for nerve tissue engineering.

The purpose of this study was to fabricate a conductive aligned nanofibrous substrate and evaluate its suitability and cytocompatibility with neural cells for nerve tissue engineering purposes. In order to reach these goals, we first used electrospinning to fabricate single-walled carbon-nanotube (SWCNT) incorporated poly(L-lactic acid) (PLLA) nanofibrous scaffolds and then assessed its cytocompatibility with olfactory ensheathing glial cells (OEC). The plasma treated scaffolds were characterized using scanning electron microscopy and water contact angle. OECs were isolated from olfactory bulb of GFP Sprague-Dawley rats and characterized using OEC specific markers via immunocytochemistry and flow cytometery. The cytocompatibility of the conductive aligned nano-featured scaffold was assessed using microscopy and MTT assay. We indicate that doping of PLLA polymer with SWCNT can augment the aligned nanosized substrate with conductivity, making it favorable for nerve tissue engineering. Our results demonstrated that SWCNT/PLLA composite scaffold promote the adhesion, growth, survival and proliferation of OEC. Regarding the ideal physical, topographical and electrical properties of the scaffold and the neurotrophic and migratory features of the OECs, we suggest this scaffold and the cell/scaffold construct as a promising platform for cell delivery to neural defects in nerve tissue engineering approaches.

Diaphragm Repair with a Novel Cross-Linked Collagen Biomaterial in a Growing Rabbit Model.

BackgroundNeonates with congenital diaphragmatic hernia and large defects often require patch closure. Acellular collagen matrices (ACM) have been suggested as an alternative to synthetic durable patches as they are remodeled by the host or could also be used for tissue engineering purposes.Materials and Methods2.0x1.0 cm diaphragmatic defects were created in 6-weeks old New-Zealand white rabbits. We compared reconstruction with a purpose-designed cross-linked ACM (Matricel) to 4-layer non-cross-linked small intestinal submucosa (SIS) and a 1-layer synthetic Dual Mesh (Gore-Tex). Unoperated animals or animals undergoing primary closure (4/0 polyglecaprone) served as age-matched controls. 60 (n = 25) resp. 90 (n = 17) days later, animals underwent chest x-ray and obduction for gross examination of explants, scoring of adhesion and inflammatory response. Also, uniaxial tensiometry was done, comparing explants to contralateral native diaphragmatic tissue.ResultsOverall weight nearly doubled from 1,554±242 g at surgery to 2,837±265 g at obduction (+84%). X-rays did show rare elevation of the left diaphragm (SIS = 1, Gore-Tex = 1, unoperated control = 1), but no herniation of abdominal organs. 56% of SIS and 10% of Matricel patches degraded with visceral bulging in four (SIS = 3, Matricel = 1). Adhesion scores were limited: 0.5 (Matricel) to 1 (SIS, Gore-Tex) to the left lung (p = 0.008) and 2.5 (Gore-Tex), 3 (SIS) and 4 (Matricel) to the liver (p<0.0001). Tensiometry revealed a reduced bursting strength but normal compliance for SIS. Compliance was reduced in Matricel and Gore-Tex (p<0.01). Inflammatory response was characterized by a more polymorphonuclear cell (SIS) resp. macrophage (Matricel) type of infiltrate (p<0.05). Fibrosis was similar for all groups, except there was less mature collagen deposited to Gore-Tex implants (p<0.05).ConclusionsMatricel induced a macrophage-dominated inflammatory response, more adhesions, had appropriate strength but a lesser compliance compared to native tissue. The herein investigated ACM is not a viable option for CDH repair.

Effect of Intensified Decellularization of Equine Carotid Arteries on Scaffold Biomechanics and Cytotoxicity

Decellularized equine carotid arteries (dEAC) are suggested to represent an alternative for alloplastic vascular grafts in haemodialysis patients to achieve vascular access. Recently it was shown that intensified detergent treatment completely removed cellular components from dEAC and thereby significantly reduced matrix immunogenicity. However, detergents may also affect matrix composition and stability and render scaffolds cytotoxic. Therefore, intensively decellularized carotids (int-dEAC) were now evaluated for their biomechanical characteristics (suture retention strength, burst pressure and circumferential compliance at arterial and venous systolic and diastolic pressure), matrix components (collagen and glycosaminoglycan content) and indirect and direct cytotoxicity (WST-8 assay and endothelial cell seeding) and compared with native (n-EAC) and conventionally decellularized carotids (con-dEAC). Both decellularization protocols comparably reduced matrix compliance (venous pressure compliance: 32.2 and 27.4% of n-EAC; p < 0.01 and arterial pressure compliance: 26.8 and 23.7% of n-EAC, p < 0.01) but had no effect on suture retention strength and burst pressure. Matrix characterization revealed unchanged collagen contents but a 39.0% (con-dEAC) and 26.4% (int-dEAC, p < 0.01) reduction of glycosaminoglycans, respectively. Cytotoxicity was not observed in either dEAC matrix which was also displayed by an intact endothelial lining after seeding. Thus, even intensified decellularization generates matrix scaffolds highly suitable for vascular tissue engineering purposes, e.g., the generation of haemodialysis shunts.

Effect of Intensified Decellularization of Equine Carotid Arteries on Scaffold Biomechanics and Cytotoxicity.

Decellularized equine carotid arteries (dEAC) are suggested to represent an alternative for alloplastic vascular grafts in haemodialysis patients to achieve vascular access. Recently it was shown that intensified detergent treatment completely removed cellular components from dEAC and thereby significantly reduced matrix immunogenicity. However, detergents may also affect matrix composition and stability and render scaffolds cytotoxic. Therefore, intensively decellularized carotids (int-dEAC) were now evaluated for their biomechanical characteristics (suture retention strength, burst pressure and circumferential compliance at arterial and venous systolic and diastolic pressure), matrix components (collagen and glycosaminoglycan content) and indirect and direct cytotoxicity (WST-8 assay and endothelial cell seeding) and compared with native (n-EAC) and conventionally decellularized carotids (con-dEAC). Both decellularization protocols comparably reduced matrix compliance (venous pressure compliance: 32.2 and 27.4% of n-EAC; p < 0.01 and arterial pressure compliance: 26.8 and 23.7% of n-EAC, p < 0.01) but had no effect on suture retention strength and burst pressure. Matrix characterization revealed unchanged collagen contents but a 39.0% (con-dEAC) and 26.4% (int-dEAC, p < 0.01) reduction of glycosaminoglycans, respectively. Cytotoxicity was not observed in either dEAC matrix which was also displayed by an intact endothelial lining after seeding. Thus, even intensified decellularization generates matrix scaffolds highly suitable for vascular tissue engineering purposes, e.g., the generation of haemodialysis shunts.

Electrospun nanofiber scaffolds with gradations in fiber organization.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.