Bilayered Construct Research Articles

Formulation and Characterization of Silk Fibroin Films as a Scaffold for Adipose-Derived Stem Cells in Skin Tissue Engineering

Skin substitutes are epidermal, dermal or complete bilayered constructs, composed by natural or synthetic scaffolds and by adherent cells such as fibroblasts, keratinocytes or mesenchymal stem cells. Silk fibroin is a promising polymer to realize scaffolds, since it is biocompatible, biodegradable, and exhibits excellent mechanical properties in terms of tensile strength. Moreover, fibroin can be added of others components in order to modify the biomaterial properties for the purpose. The aim of this work is to prepare silk fibroin films for adipose-derived stem cell (ADSCs) culture as a novel feeder layer for skin tissue engineering. Pectin has been added to promote the protein conformational transition and construct strength, while glycerol as plasticizer, providing biomaterial flexibility. Eighteen formulations were prepared by casting method using fibroin, pectin (range 1-10% w/w), and glycerol (range 0-20% w/w); films were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry assay, to select the optimal composition. A stable fibroin conformation was obtained using 6% w/w pectin, and the best mechanical properties were obtained using 12% w/w glycerol. Films were sterilized, and human ADSCs were seeded and cultured for 15 days. Cells adhere to the support assuming a fibroblastic-like shape and reaching confluence. The ultrastructural analysis evidences typical active-cell features and adhesion structures that promote cell anchorage to the film, thus developing a multilayered cell structure. This construct could be advantageously employed in cutaneous wound healing or where the use of ADSCs scaffold is indicated either in human or veterinary field.

Stratified scaffolds for osteochondral tissue engineering applications: Electrospun PDLLA nanofibre coated Bioglass®-derived foams

This work focuses on designing bilayered constructs by combining electrospun poly-DL-Lactide (PDLLA) fibers and Bioglass®-derived scaffolds for development of osteochondral tissue replacement materials. Electrospinning was carried out using a solution of 5 wt/v% PDLLA in dimethyl carbonate. The PDLLA layer thickness increased from 2 to 150 µm with varying electrospinning time. Bioactivity studies in simulated body fluid showed that HA mineralization decreased as the thickness of the PDLLA layer increased. A preliminary in vitro study using chondrocyte cells (ATDC5) showed that cells attached, proliferated and migrated into the fibrous network, confirming the potential applicability of the bilayered scaffolds in osteochondral defect regeneration.

Differential Keratin Expression During Epiboly in a Wound Model of Bioengineered Skin and in Human Chronic Wounds

Epiboly represents the process by which keratinocytes migrate to envelop a surface. The authors have been investigating a living bilayered skin construct (BSC) that is used in the treatment of lower extremity wounds due to venous insufficiency and diabetes. The construct demonstrates epiboly after injury and incubation in vitro, and this model may be useful for studying epidermal migration and the process of skin maturation. Punch biopsies of the construct in vitro were cultured and immunostained for specific keratins at baseline and at 24 to 72 hours. For comparison, skin biopsy specimens from human chronic venous ulcers and acute healing wounds were similarly processed. The authors found that K1 and K10 were fully expressed in the epidermis of the fully epibolized surface on BSC. K1 was also present in the migrating edge of specimens, whereas K10 was not detectable. K16 and K6 were evident in normal skin and the epibolized area of the construct; K6 expression was very prominent in the migrating edge. Importantly, K17 was distinctly limited to the epibolized surface and the migrating edge, and its expression was very similar to that observed in healing human wounds. In conclusion, differential expression of keratins in this epiboly model closely reflects in vivo studies and supports keratin specificity in the processes of migration and differentiation of new epidermis. Therefore, these findings provide further and important validity for the study of epithelialization and the hope of developing prognostic markers for venous ulcer healing.

Demonstration of a convergent approach to UV-polymerizable lipids bisDenPC and bisSorbPC

Lipids containing UV-polymerizable diene moieties, such as BisDenPC 1 and BisSorbPC 2, have been extremely useful for the construction of micelles and lipid bilayers. The published syntheses have yielded lipids with only a trimethylamine head group. We have improved the syntheses of these monomers by a convergent method employing the Chabrier reaction of trimethylamine with a cyclic phospholane. This method can be extended to a variety of dialkylamine derivatives.

Smectic Demixing in the Phase Behavior and Self-Assembly of a Hydrogen-Bonded Polymer with Mesogenic Side Chains

We present a detailed study of the structure and properties of a supramolecular complex formed via hydrogen bond association between poly(acrylic acid) chains and an imidazole-terminated biphenyl mesogen. The system exhibits a rich phase behavior as a function of temperature and stoichiometry, expressed as the molar ratio S between the number of mesogens and binding sites present. Smectic mesophases are formed for all S ≥ 0.033, a surprisingly small number. The dependence of the characteristic length scale of the mesophase on stoichiometry does not follow the expected 1-D swelling law. At low stoichiometries, S ≤ 0.2, the system exhibits little or no change in structure up to temperatures as high as 200 °C, beyond which changes become temperature irreversible. In contrast, at higher S, the system features complex thermally driven transitions among tilted monolayer and bilayer arrangements and complete, reversible isotropization of the system at elevated temperatures. Over a limited range of temperatures and compositions, a supramolecular length scale emerges that is well beyond the upper limit imposed by a bilayer construct and thus cannot be accounted for within the conventional paradigm. Binding isotherms reveal that the polymer has a limited capacity for the ligand with saturation occurring for S ≥ 0.33. These results suggest that the common assumption of homogeneously distributed tightly bound ligands with layer-like phase separation from the polymer backbone do not apply in this system over all compositions. The anomalous phase display is consistent with demixing between polymer rich and polymer poor domains due to the presence of excess unassociated mesogen, which can act as a solvent for the system.

Approach for Fabricating Tissue Engineered Vascular Grafts with Stable Endothelialization

A major roadblock in the development of tissue engineered vascular grafts (TEVGs) is achieving construct endothelialization that is stable under physiological stresses. The aim of the current study was to validate an approach for generating a mechanically stable layer of endothelial cells (ECs) in the lumen of TEVGs. To accomplish this goal, a unique method was developed to fabricate a thin EC layer using poly(ethylene glycol) diacrylate (PEGDA) as an intercellular "cementing" agent. This EC layer was subsequently bonded to the lumen of a tubular scaffold to generate a bi-layered construct. The viability of bovine aortic endothelial cells (BAECs) through the "cementing" process was assessed. "Cemented" EC layer expression of desired phenotypic markers (AcLDL uptake, VE-cadherin, eNOS, PECAM-1) as well as of injury-associated markers (E-selectin, SM22alpha) was also examined. These studies indicated that the "cementing" process allowed ECs to maintain high viability and expression of mature EC markers while not significantly stimulating primary injury pathways. Finally, the stability of the "cemented" EC layers under abrupt application of high shear pulsatile flow (approximately 11 dyn/cm(2), P (avg) approximately 95 mmHg, DeltaP approximately 20 mmHg) was evaluated and compared to that of conventionally "seeded" EC layers. Whereas the "cemented" ECs remained fully intact following 48 h of pulsatile flow, the "seeded" EC layers delaminated after less than 1 h of flow. Furthermore, the ability to extend this approach to degradable PEGDA "cements" permissive of cell elongation was demonstrated. Combined, these results validate an approach for fabricating bi-layered TEVGs with stable endothelialization.

Electrically Addressable, Biologically Relevant Surface-Supported Bilayers

The assembly of electrically addressable, planar supported bilayers composed of biologically relevant lipids, such as those used in vesicular systems, will greatly enhance the experimental capabilities in membrane and membrane protein research. Here we assess the electrical properties of bilayers composed of a wide range of physiologically relevant lipids and lipid combinations. We demonstrate that robust, biologically relevant, planar supported bilayers with high resistance composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 25 mol % cholesterol can be constructed with high reproducibility. Furthermore, to enable studies of pore-forming peptides, which are commonly cationic, we demonstrate the construction of bilayers with biologically relevant outer leaflets incorporating up to 10 mol % negatively charged lipids. Unique features of the platform are that (1) the substrate is commercially available, atomically smooth, single-crystal silicon, (2) the polymer cushion allows for the natural incorporation of membrane proteins, and (3) the platform is highly reproducible.

Effects of TGF-β3 and preculture period of osteogenic cells on the chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in a bilayered hydrogel composite

In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) and gelatin microparticles (MPs) were used to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-beta3 (TGF-beta3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-beta3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-beta3 significantly stimulated chondrogenic differentiation of MSCs. In addition, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-beta3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-beta3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites which mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.

Functional testing of a tissue-engineered vocal fold cover replacement

Objectives Tissue engineering may provide a treatment for severe vocal fold scars. This study quantifies mechanical properties and demonstrates vibration of a tissue-engineered vocal fold cover replacement. Methods Tissue-engineered constructs were produced from fibrin and adipose-derived stem cells. Optimized bilayered constructs contained epithelial and mesenchymal cell phenotypes in a stratified geometry. For comparison, homogeneous constructs did not have epithelial differentiation. Elastic modulus was determined using indentation. Immunohistochemical labeling for type I collagen was performed. A bilayered construct was also tested in phonation in an excised larynx model. Results Bilayered vocal fold cover replacements had indentation moduli similar to human vocal fold covers (mean construct modulus 6.8 kPa). Collagen deposition occurred in the middle of the construct. Homogeneous constructs had a mean modulus of 8.3 kPa, and collagen was concentrated at the surface. An excised larynx with unilateral vocal fold cover replacement phonated and exhibited mucosal waves at physiologic airflow. Conclusion Bilayered tissue-engineered constructs were produced that exhibited indentation modulus, microstructure, and vibration similar to that exhibited by human vocal fold covers.

In vitro generation of an osteochondral construct using injectable hydrogel composites encapsulating rabbit marrow mesenchymal stem cells

Injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized to fabricate a bilayered osteochondral construct consisting of a chondrogenic layer and an osteogenic layer, and to investigate the differentiation of rabbit marrow mesenchymal stem cells (MSCs) encapsulated in both layers in vitro. The results showed that MSCs in the chondrogenic layer were able to undergo chondrogenic differentiation, especially in the presence of TGF-β1-loaded MPs. In the osteogenic layer, cells maintained their osteoblastic phenotype. Although calcium deposition in the osteogenic layer was limited, cells in the osteogenic layer significantly enhanced chondrogenic differentiation of MSCs in the chondrogenic layer. The greatest effect was observed when MSCs were encapsulated with TGF-β1-loaded MPs and cultured with osteogenic cells in the bilayered constructs. Overall, this study demonstrates the fabrication of bilayered hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers.

Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture

BackgroundPoly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion.ResultsA systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip.ConclusionThe obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Different Apolipoproteins Impact Nanolipoprotein Particle Formation

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.

Bi-layered constructs based on poly( l-lactic acid) and starch for tissue engineering of osteochondral defects

Articular cartilage has a limited capacity to repair itself, and conventional therapeutic approaches have shown to have limited success as they are deficient and inconsistent in long-term repair. Tissue engineering has shown to be an alternative route to regenerate articular defects. In this work, new bi-layered scaffolds are developed in order to enhance the integration between the engineered cartilage tissue and the corresponding subchondral bone. The concept includes the use of a common polymer in both sides, poly( l-lactic acid), PLLA, to increase the bonding between them, and the use of compression moulding followed by particle leaching to process porous scaffolds with controllable porosities. A compact layer could be observed between the two layers that could be useful for independent cell culturing of the developed osteochondral constructs. A blend of starch and PLLA was used in the cartilage side, which was found to possess adequate hydration capability. For the bone region, where more stiffness and strength was required, PLLA reinforced with hydroxyapatite was used. Preliminary bioactivity tests demonstrated that the bone-layer could induce the formation of a calcium–phosphate layer in vitro, whereas the cartilage layer does not exhibit the ability for calcification.

Wound bed score and its correlation with healing of chronic wounds

Adequate wound bed preparation is essential for healing of leg ulcers, and consists of controlling exudate and edema, decreasing the bacterial burden, promoting healthy granulation tissue, and removing necrotic tissue. Currently, there is no classification system for wound bed preparation that has predictive value. Based on past work and the authors' experience, we have now developed and tested a new classification system that scores the following parameters: healing edges (wound edge effect), presence of eschar, greatest wound depth/granulation tissue, amount of exudate amount, edema, peri-wound dermatitis, peri-wound callus and or fibrosis, and a pink/red wound bed. Each parameter receives a score from 0 (worst score) to 2 (best score), and all the parameter scores are added for a total score. Each wound can have a maximum score of 16 (the best score possible), to a minimum score of 0 (the worst score possible). We used this wound bed score (WBS) system in a study of 177 patients with venous ulcers who had been prospectively treated with and randomized to either conventional therapy (compression alone) or a living bilayered skin construct (BSC). We evaluated serial photographs at baseline to determine whether the results would be predictive of complete wound closure and could validate the WBS. We found that wounds that ultimately achieved full closure had a statistically significant higher WBS than those that did not heal (p = 0.0012). This was also true when separating wounds by treatment modality: standard therapy (p = 0.044) and treatment with a BSC (p = 0.011). When dividing the WBS in the following quartile groups: scores 4-10, 10-12, 12-13, and 13-16, the percentage of healed wounds correlated with the WBS (p = 0.0008). For all wounds, a one unit increase in total WBS resulted on average in a 22.8% increase in odds of healing (OR = 1.228). This WBS seems to have validity in predicting complete wound closure in wounds treated with either standard therapy or advanced modalities, such as BSC. If confirmed and widely adopted in this and other types of wounds, it could be a useful tool in both the clinical and research setting.

Artificial Skin as a Valuable Adjunct to Surgical Treatment of a Large Squamous Cell Carcinoma in a Patient with Epidermolysis Bullosa

Among tissue-engineered skins, two bilayered cellular constructs and one cryopreserved dermal substitute have been approved for the treatment of epidermolysis bullosa. Nevertheless, the application of artificial skin technology to surgical treatment of squamous cell carcinomas in a patient with epidermolysis bullosa has never been reported. To reconstruct the large defect remaining after squamous cell carcinoma excision in a patient with dominantly inherited dystrophic epidermolysis bullosa. To apply a 10 x 15 cm Integra sheet (Integral Life-sciences Corporation, Plainsboro, NJ, USA) (an acellular collagen matrix coated with a thin polysiloxane elastomer) to the excised area and 3 weeks later to cover the Integra sheet with an ultrathin meshed skin graft. The graft take was complete, and the donor site totally regenerated, except for three small bullae at 7 weeks postoperatively. Integra offers the advantage of filling huge defects with its dermal layer of collagen fibers and provides an optimal graft bed. This first step makes it possible to use very thin grafts 3 weeks later.

Bioactive Wound Healing, Bioaesthetics and Biosurgery: Three Pillars of Product Development

Tissue regeneration specialist company Organogenesis Inc. was one of the first biotech companies formed. Incorporated in 1985, the company was originally a spin-off from a research program at MIT. For the first 10-15 years, Organogenesis was heavily research based, but then gradually moved into development. The company's flagship product is Apligraf--a living, bilayered skin construct with two FDA-approved indications: diabetic foot ulcers and venous leg ulcers. As Apligraf neared the market, it was necessary to 'graft' a manufacturing capability onto the company. As a consequence the company moved south from Massachusetts's cradle of biotechnology to Canton, MA, USA. Having experienced many of the highs and lows that characterize the biotech industry, the company is now consolidating its position as a center of expertise in commercializing living, cell-based products. The company has now built a sales, marketing and reimbursement team with the unique skill set to integrate novel technology into the US healthcare system. President & Chief Executive Officer Geoff MacKay takes great pride in the leading role that Organogenesis is playing in ushering in the field of tissue regeneration. Here, he discusses with Regenerative Medicine's Elisa Manzotti the 'three pillars' of the Organogenesis pipeline: bioactive wound healing, bioaesthetics and biosurgery. He focuses both on the rewards, and the trials and tribulations, of the commercialization of living cell-based technology.

Safety and Efficacy of a Bilayered Skin Construct in Full-Thickness Surgical Wounds

This was a prospective, randomized, controlled clinical trial assessing the safety and efficacy of a living bilayered skin construct (BSC; Organogenesis, Canton, MA) in treating full-thickness surgical excision wounds. We enrolled 31 patients needing excision of a non-melanoma skin cancer. The patients consisted of 18 females and 13 males, with an average age of 67 years (range: 44 to 84 years). Patients were randomized to either receive a single application of BSC or to heal by secondary intention. Endpoints to assess efficacy included time to complete wound closure, intensity and duration of post-operative pain, cosmetic appearance, patient satisfaction, and quality of the healed wound. Endpoints to assess safety included infection at the wound site and rejection of the BSC. Findings indicate that BSC is safe in the post-operative treatment of acute surgical wounds for removal of non-melanoma skin cancer. The data also suggest that BSC may facilitate management by decreasing post-operative pain. It is unclear whether or not BSC decreases healing time of acute wounds or results in a better cosmetic outcome.

Artificial Skin as a Valuable Adjunct to Surgical Treatment of a Large Squamous Cell Carcinoma in a Patient with Epidermolysis Bullosa

Among tissue-engineered skins, two bilayered cellular constructs and one cryopreserved dermal substitute have been approved for the treatment of epidermolysis bullosa. Nevertheless, the application of artificial skin technology to surgical treatment of squamous cell carcinomas in a patient with epidermolysis bullosa has never been reported. Objective To reconstruct the large defect remaining after squamous cell carcinoma excision in a patient with dominantly inherited dystrophic epidermolysis bullosa. Methods To apply a 10 × 15 cm Integra sheet (Integral Lifesciences Corporation, Plainsboro, NJ, USA) (an acellular collagen matrix coated with a thin polysiloxane elastomer) to the excised area and 3 weeks later to cover the Integra sheet with an ultrathin meshed skin graft. Results The graft take was complete, and the donor site totally regenerated, except for three small bullae at 7 weeks postoperatively. Conclusion Integra offers the advantage of filling huge defects with its dermal layer of collagen fibers and provides an optimal graft bed. This first step makes it possible to use very thin grafts 3 weeks later.

Bioengineered bilayered skin construct preservation using epiboly as a measure of construct functionality

Bioengineered bilayered skin construct preservation using epiboly as a measure of construct functionality