Showing papers in "Journal of Biomedical Materials Research Part A in 2013"

3D bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels.

Abstract: Heart valve disease is a serious and growing public health problem for which prosthetic replacement is most commonly indicated. Current prosthetic devices are inadequate for younger adults and growing children. Tissue engineered living aortic valve conduits have potential for remodeling, regeneration, and growth, but fabricating natural anatomical complexity with cellular heterogeneity remain challenging. In the current study, we implement 3D bioprinting to fabricate living alginate/gelatin hydrogel valve conduits with anatomical architecture and direct incorporation of dual cell types in a regionally constrained manner. Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced modulus, ultimate strength, and peak strain reducing slightly over 7-day culture, while the tensile biomechanics of cell-laden hydrogels were maintained. Aortic valve conduits were successfully bioprinted with direct encapsulation of SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4 ± 3.4% for SMC and 83.2 ± 4.0% for VIC) within 3D printed tissues. Encapsulated SMC expressed elevated alpha-smooth muscle actin, while VIC expressed elevated vimentin. These results demonstrate that anatomically complex, heterogeneously encapsulated aortic valve hydrogel conduits can be fabricated with 3D bioprinting.

Evaluation of hydrogels for bio-printing applications.

Abstract: In the United States alone, there are approximately 500,000 burn injuries that require medical treatment every year. Limitations of current treatments necessitate the development of new methods that can be applied quicker, result in faster wound regeneration, and yield skin that is cosmetically similar to undamaged skin. The development of new hydrogel biomaterials and bioprinting deposition technologies has provided a platform to address this need. Herein we evaluated characteristics of twelve hydrogels to determine their suitability for bioprinting applications. We chose hydrogels that are either commercially available, or are commonly used for research purposes. We evaluated specific hydrogel properties relevant to bioprinting applications, specifically; gelation time, swelling or contraction, stability, biocompatibility and printability. Further, we described regulatory, commercial and financial aspects of each of the hydrogels. While many of the hydrogels screened may exhibit characteristics suitable for other applications, UV-crosslinked Extracel, a hyaluronic acid-based hydrogel, had many of the desired properties for our bioprinting application. Taken together with commercial availability, shelf life, potential for regulatory approval and ease of use, these materials hold the potential to be further developed into fast and effective wound healing treatments.

On the biodegradability, mechanical behavior, and cytocompatibility of amorphous Mg72Zn23Ca5and crystalline Mg70Zn23Ca5Pd2alloys as temporary implant materials

Abstract: The evolution of microstructure and mechanical properties of almost fully amorphous Mg72Zn23Ca5 and crystalline Mg70Zn23Ca5Pd2 alloys during immersion in Hank's balanced salt solution (HBSS), as well as their cytocompatibility, are investigated in order to assess the feasibility of both materials as biodegradable implants. Though the crystalline Mg70Zn23Ca5Pd2 sample shows lower wettability and more positive corrosion potential, this sample degrades much faster upon incubation in HBSS as a consequence of the formation of micro-galvanic couples between the nobler Pd-rich dendrites and the surrounding phases. After 22-h immersion, the concentration of Mg ions in the HBSS medium containing the Mg70Zn23Ca5Pd2 sample is six times larger than for Mg72Zn23Ca5. Due to the Zn enrichment and the incipient porosity, the mechanical properties of the Mg72Zn23Ca5 sample improve within the first stages of biodegradation (i.e., hardness increases while the Young's modulus decreases, thus rendering an enhanced wear resistance). Cytocompatibility studies reveal that neither Mg72Zn23Ca5 nor Mg70Zn23Ca5Pd2 are cytotoxic, although preosteoblast cell adhesion is to some extent precluded, particularly onto the surface of Mg70Zn23Ca5Pd2, because of the relatively high hydrophobicity. Because of their outstanding properties and their time-evolution, the use of the Pd-free alloy in temporary implants such as screws, stents, and sutures is envisioned.

Nanostructured scaffolds for bone tissue engineering.

Abstract: It has been demonstrated that nanostructured materials, compared with conventional materials, may promote greater amounts of specific protein interactions, thereby more efficiently stimulating new bone formation. It has also been indicated that, when features or ingredients of scaffolds are nanoscaled, a variety of interactions can be stimulated at the cellular level. Some of those interactions induce favorable cellular functions while others may leads to toxicity. This review presents the mechanism of interactions between nanoscaled materials and cells and focuses on the current research status of nanostructured scaffolds for bone tissue engineering. Firstly, the main requirements for bone tissue engineering scaffolds were discussed. Then, the mechanism by which nanoscaled materials promote new bone formation was explained, following which the current research status of main types of nanostructured scaffolds for bone tissue engineering was reviewed and discussed.

Macrophages – Key Cells in the Response to Wear Debris from Joint Replacements

Abstract: The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of pro-inflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented.

Cell response of anodized nanotubes on titanium and titanium alloys.

Abstract: Titanium and titanium alloy implants that have been demonstrated to be more biocompatible than other metallic implant materials, such as Co-Cr alloys and stainless steels, must also be accepted by bone cells, bonding with and growing on them to prevent loosening. Highly ordered nanoporous arrays of titanium dioxide that form on titanium surface by anodic oxidation are receiving increasing research interest due to their effectiveness in promoting osseointegration. The response of bone cells to implant materials depends on the topography, physicochemistry, mechanics, and electronics of the implant surface and this influences cell behavior, such as adhesion, proliferation, shape, migration, survival, and differentiation; for example the existing anions on the surface of a titanium implant make it negative and this affects the interaction with negative fibronectin (FN). Although optimal nanosize of reproducible titania nanotubes has not been reported due to different protocols used in studies, cell response was more sensitive to titania nanotubes with nanometer diameter and interspace. By annealing, amorphous TiO2 nanotubes change to a crystalline form and become more hydrophilic, resulting in an encouraging effect on cell behavior. The crystalline size and thickness of the bone-like apatite that forms on the titania nanotubes after implantation are also affected by the diameter and shape. This review describes how changes in nanotube morphologies, such as the tube diameter, the thickness of the nanotube layer, and the crystalline structure, influence the response of cells.

Bioactive effects of graphene oxide cell culture substratum on structure and function of human adipose‐derived stem cells

Abstract: Nanoscale topography of artificial substrates can greatly influence the fate of stem cells including adhesion, proliferation, and differentiation. Thus the design and manipulation of nanoscale stem cell culture platforms or scaffolds are of great importance as a strategy in stem cell and tissue engineering applications. In this report, we propose that a graphene oxide (GO) film is an efficient platform for modulating structure and function of human adipose-derived stem cells (hASCs). Using a self-assembly method, we successfully coated GO on glass for fabricating GO films. The hASCs grown on the GO films showed increased adhesion, indicated by a large number of focal adhesions, and higher correlation between the orientations of actin filaments and vinculin bands compared to hASCs grown on the glass (uncoated GO substrate). It was also found that the GO films showed the stronger affinity for hASCs than the glass. In addition, the GO film proved to be a suitable environment for the time-dependent viability of hASCs. The enhanced differentiation of hASCs included osteogenesis, adipogenesis, and epithelial genesis, while chondrogenic differentiation of hASCs was decreased, compared to tissue culture polystyrene as a control substrate. The data obtained here collectively demonstrates that the GO film is an efficient substratum for the adhesion, proliferation, and differentiation of hASCs.

Nano‐hydroxyapatite‐coated PEEK implants: A pilot study in rabbit bone

Abstract: Osseointegration of surface-modified polyetheretherketone (PEEK) implants was studied in vivo. A total of 18 cylinder-shaped PEEK implants were inserted in the femurs of nine New Zealand rabbits; half were coated with nanocrystalline hydroxyapatite (nanoHA) and half were uncoated controls. Healing time was 6 weeks. Samples were retrieved with the implant and surrounding tissue, processed to cut and ground sections, and analyzed histomorphometrically. The implant surfaces were analyzed with optical interferometry, scanning electron microscopy (SEM), atomic force microscopy, and X-ray photoelectron spectroscopy (XPS). NanoHA-coated PEEK surfaces had lower height deviation (Sa) than controls [mean ± SD: 0.41 μm (± 0.14) vs. 0.96 μm (± 0.28)]. SEM images showed the nanoHA crystals as a thin layer on the polymer surface. XPS analysis of the coated implants showed a Ca/P ratio of 1.67. Histomorphometry indicated that the nanoHA-coated implants had more bone-to-implant contact [16% (± 4.7) vs. 13% (± 9.3)] and more bone area [52% (± 9.5) vs. 45% (± 11.9)]. We found no difference between smooth nanoHA-coated cylinder-shaped PEEK implants and uncoated controls. However, higher mean bone-to-implant contact indicated better osseointegration in the coated implants than in the uncoated controls. The large number of lost implants was interpreted as a lack of primary stability due to implant design.

Dental implants from functionally graded materials

Abstract: Functionally graded material (FGM) is a heterogeneous composite material including a number of constituents that exhibit a compositional gradient from one surface of the material to the other subsequently, resulting in a material with continuously varying properties in the thickness direction. FGMs are gaining attention for biomedical applications, especially for implants, owing to their reported superior composition. Dental implants can be functionally graded to create an optimized mechanical behavior and achieve the intended biocompatibility and osseointegration improvement. This review presents a comprehensive summary of biomaterials and manufacturing techniques researchers employ throughout the world. Generally, FGM and FGM porous biomaterials are more difficult to fabricate than uniform or homogenous biomaterials. Therefore, our discussion is intended to give the readers about successful and obstacles fabrication of FGM and porous FGM in dental implants that will bring state-of-the-art technology to the bedside and develop quality of life and present standards of care.

In vitro antibacterial evaluation of sol-gel-derived Zn-, Ag-, and (Zn + Ag)-doped hydroxyapatite coatings against methicillin-resistant Staphylococcus aureus.

Abstract: Hydroxyapatite (HAp) coatings were applied using sol-gel method. Phosphor pentoxide and calcium nitrate were used as phosphorous and calcium precursors, respectively. Zinc nitrate and silver nitrate were used as substitute of calcium in HAp structure. As a base concentration, 1.5 wt %Ag and 2.5 wt %Zn were used. The weight percent of Ag was increased at 0.3 wt% and Zn content was scaled down at 0.5 wt%. Phase analysis and chemical bonds of synthesized materials were studied by XRD and FTIR. Antibacterial activity of Ag- and Zn-doped samples against methicilin-resistant Staphylococcus aureus (MRSA) were assessed by the plate-counting method. The XRD and FTIR results proved formation of HAp compound. Colony counting showed that silver and zinc ions prevent proliferation and growth of MRSA. Interestingly, co-presence of metal ions improves the antibacterial effectiveness of the coatings and the combined effect was greater than sum of the individual effects when each was administered alone. Overall, synergism between antibacterial activities of Zn(2+) and Ag(+) ions against MRSA can be suggested. Thus, cell toxicity decreases and biocompatibility increases without any decrement in antibacterial activity.

Electrospinning collagen/chitosan/poly(L-lactic acid-co-ε-caprolactone) to form a vascular graft: mechanical and biological characterization.

Abstract: For blood vessel tissue engineering, an ideal vascular graft should possess excellent biocompatibility and mechanical properties. For this study, a elastic material of poly (L-lactic acid-co-e-caprolactone) (P(LLA-CL)), collagen and chitosan blended scaffold at different ratios were fabricated by electrospinning. Upon fabrication, the scaffolds were evaluated to determine the tensile strength, burst pressure, and dynamic compliance. In addition, the contact angle and endothelial cell proliferation on the scaffolds were evaluated to demonstrate the structures potential to serve as a vascular prosthetic capable of in situ regeneration. The collagen/chitosan/P(LLA-CL) scaffold with the ratio of 20:5:75 reached the highest tensile strength with the value of 16.9 MPa, and it was elastic with strain at break values of ~112%, elastic modulus of 10.3 MPa. The burst pressure strength of the scaffold was greater than 3365 mmHg and compliance value was 0.7%/100 mmHg. Endothelial cells proliferation was significantly increased on the blended scaffolds versus the P(LLA-CL). Meanwhile, the endothelial cells were more adherent based on the increase in the degree of cell spreading on the surface of collagen/chitosan/P(LLA-CL) scaffolds. Such blended scaffold especially with the ratio of 20:5:75 thus has the potential for vascular graft applications.

Preparation and characterization of collagen-nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications.

Abstract: Recent efforts of bone repair focus on development of porous scaffolds for cell adhesion and proliferation Collagen-nanohydroxyapatite (HA) scaffolds (70:30; 50:50; and 30:70 mass percentage) were produced by cryogelation technique using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide as crosslinking agents A pure collagen scaffold was used as control Morphology analysis revealed that all cryogels had highly porous structure with interconnective porosity and the nanoHA aggregates were randomly dispersed throughout the scaffold structure Chemical analysis showed the presence of all major peaks related to collagen and HA in the biocomposites and indicated possible interaction between nanoHA aggregates and collagen molecules Porosity analysis revealed an enhancement in the surface area as the nanoHA percentage increased in the collagen structure The biocomposites showed improved mechanical properties as the nanoHA content increased in the scaffold As expected, the swelling capacity decreased with the increase of nanoHA content In vitro studies with osteoblasts cells showed that they were able to attach and spread in all cryogels surfaces The presence of collagen-nanoHA biocomposites resulted in higher overall cellular proliferation compared to pure collagen scaffold A statistically significant difference between collagen and collagen-nanoHA cryogels was observed after 21 day of cell culture These innovative collagen-nanoHA cryogels could have potentially appealing application as scaffolds for bone regeneration © 2012 Wiley Periodicals, Inc J Biomed Mater Res Part A, 2013

Processing and evaluation of bioactive coatings on polymeric implants

Abstract: Polyetheretherketone (PEEK) is a high-performance polymer with advantages over metallic biomaterials for application in spinal implants. In this study, hydroxyapatite (HA) coatings were deposited onto PEEK substrates using radio-frequency magnetron sputtering for the purpose of improving bioactivity. An intermediate coating layer of yttria-stabilized zirconia (YSZ) was first deposited onto the PEEK substrates to provide heat shielding during subsequent post-deposition heat treatment to prevent degradation of PEEK substrates and coating/substrate interface. Plasma activation of the PEEK substrate surfaces before deposition resulted in a significant increase in coating adhesion strength. Post-deposition heat treatments of microwave and hydrothermal annealing were studied with the goal of forming crystalline HA without the use of high temperatures required in conventional annealing. Microstructural and compositional analyses by scanning electron microscopy (SEM) and X-ray diffraction revealed that the YSZ layer exhibited a crystalline structure as-deposited, with columnar grains oriented along the growth direction, whereas the HA layer was shown to be amorphous as-deposited. After microwave annealing, the HA coating exhibited a columnar crystalline microstructure, similar to that of the underlying YSZ crystalline layer; XRD analysis confirmed a crystalline HA phase in the coating. It is suggested that the existence of the crystalline YSZ layer aids in the formation of the HA layer upon heating, possibly lowering the activation energy for crystallization by providing nucleation sites for HA grain formation. Cell culture tests showed a significant increase in initial cell attachment and growth on the microwave-annealed coatings, compared with uncoated PEEK and amorphous HA surfaces.

The effect of surface roughness on RAW 264.7 macrophage phenotype.

Abstract: Monocyte-derived cells, including macrophages and foreign body giant cells, can determine the performance of implanted devices. Upon contact with biomaterials, macrophages can be activated into a classic inflammatory (M1) or wound-healing (M2) phenotype. Previously, we showed that high macrophage density on rough SLA implants was associated with early bone formation. This study examined a possible mechanism, namely, surface roughness activation of macrophages to the M2 phenotype to enhance bone formation on the SLA surface. RAW 264.7 macrophages were seeded on SLA or smooth (Po) epoxy substrates and the expression of the M1 and M2 specific markers, NOS2 and Arg-1 measured by qPCR on days 1, 3, and 5. Additionally, secretion of inflammation-associated cytokines and chemokines was studied by antibody arrays and ELISAs. Controls included RAW 264.7 macrophages primed into the M1 or M2 phenotypes by LPS/IFN-γ and IL-4, respectively. Rough SLA surfaces did not activate Arg-1 and NOS2 expression, but relative to Po surfaces MCP-1 and MIP-1α were upregulated after 5 days, whereas the secretion of the M1-associated chemokine IP-10 was lowered. RAW 264.7 macrophages on the SLA surface thus adopted elements of an M2-like phenotype, suggesting that when implanted the SLA surfaces may enhance wound repair. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 2679–2688, 2013.

Correlating macrophage morphology and cytokine production resulting from biomaterial contact.

Abstract: The morphological and inflammatory responses of adherent macrophages are correlated to evaluate the biocompatibility of surfaces. Monocyte-derived macrophage (MDM), THP-1, and THP-1 cells expressing GFP-actin chimeric protein were seeded onto glass, polyurethane (PU), and glass surface modified with quaternary ammonium salt functionalized chitosan (CH-Q) and hyaluronic acid (HA). Using confocal microscopy, the surface area, volume and 3D shape factor of adherent macrophages was quantified. For comparison, functional consequences of cell-surface interactions that activate macrophages and thereby elicit secretion of a proinflammatory cytokine were evaluated. Using an enzyme linked immune sorbent assay, tumor necrosis factor-alpha (TNF-α) was measured. On glass, macrophages exhibited mainly an amoeboid shape, exhibited the largest surface area, volume, and 3D shape factor and produced the most TNF-α. On PU, macrophages displayed mainly a hemispherical shape, exhibited an intermediate volume, surface area and 3D shape factor, and produced moderate TNF-α. In contrast, on CH-Q and HA surfaces, macrophages were spherical, exhibited the smallest volume, surface area, and 3D shape factor, and produced the least TNF-α. These studies begin to validate the use of GFP-actin-modified MDM as a novel tool to correlate cell morphology with inflammatory cell response.

Water-stable electrospun collagen fibers from a non-toxic solvent and crosslinking system.

Abstract: Cytocompatible and water-stable ultrafine collagen fibers were electrospun by dissolving collagen in a low corrosive ethanol–water solvent and crosslinked by citric acid (CA) with glycerol as the crosslinking extender. Conventional solvents used for electrospinning of collagen either cause denaturation or contain more than 50% salt potentially leading to poor mechanical properties and water stability of the scaffolds. Collagen scaffolds have to be modified by techniques, such as, crosslinking to overcome the limitations in strength and stability. However, the existing crosslinking methods are either cytotoxic or ineffective. In this research, a benign ethanol-water solvent system and an extender-aided CA crosslinking method were developed. The native collagen conformation was retained after electrospinning, and the dry/wet strengths and water stability of fibers were substantially enhanced after crosslinking. The crosslinked electrospun scaffolds could maintain their fibrous structure for up to 30 days in phosphate-buffered saline at 37°C. Cells exhibited better attachment and growth on the CA crosslinked collagen fibers than on the glutaraldehyde crosslinked scaffolds. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Impact of the biophysical features of a 3D gelatin microenvironment on glioblastoma malignancy

Abstract: Three-dimensional tissue engineered constructs provide a platform to examine how the local extracellular matrix (ECM) contributes to the malignancy of cancers such as human glioblastoma multiforme. Improved resolution of how local matrix biophysical features impact glioma proliferation, genomic and signal transduction paths, as well as phenotypic malignancy markers would complement recent improvements in our understanding of molecular mechanisms associated with enhanced malignancy. Here, we report the use of a gelatin methacrylate (GelMA) platform to create libraries of three-dimensional biomaterials to identify combinations of biophysical features that promote malignant phenotypes of human U87MG glioma cells. We noted key biophysical properties, namely matrix density, crosslinking density, and biodegradability, that significantly impact glioma cell morphology, proliferation, and motility. Gene expression profiles and secreted markers of increased malignancy, notably VEGF, MMP-2, MMP-9, HIF-1, and the ECM protein fibronectin, were also significantly impacted by the local biophysical environment as well as matrix-induced deficits in diffusion-mediated oxygen and nutrient biotransport. Overall, this biomaterial system provides a flexible platform to explore the role biophysical factors play in the etiology, growth, and subsequent invasive spreading of gliomas.

Investigation of potential injectable polymeric biomaterials for bone regeneration

Abstract: This article reviews the potential injectable polymeric biomaterial scaffolds currently being investigated for application in bone tissue regeneration. Two types of injectable biomaterial scaffolds are focused in this review, including injectable microspheres and injectable gels. The injectable microspheres section covers several polymeric materials, including poly(L-lactide-co-glycolide)-PLGA, poly(propylene fumarate), and chitosan. The injectable gel section covers alginate gels, hyaluronan hydrogels, poly(ethylene-glycol)-PEG hydrogels, and PEG-PLGA copolymer hydrogels. This review focuses on the effect of cellular behavior in vitro and in vivo in terms of material properties of polymers, such as biodegradation, biocompatibility, porosity, microsphere size, and cross-linking nature. Injectable polymeric biomaterials offer a major advantage for orthopedic applications by allowing the ability to use noninvasive or minimally invasive treatment methods. Therefore, combining injectable polymeric biomaterial scaffolds with cells have a significant potential to treat orthopedic bone defects, including spine fusion, and craniofacial and periodontal defects.

Novel selenium-doped hydroxyapatite coatings for biomedical applications.

Abstract: Nowadays there is a short-term need of investigating in orthopedic implants with a greater functionality, including an improved osseointegration and also antibacterial properties. The coating of metallic implants with hydroxyapatite (HA) remains to be the main proposal, but superior quality HA coatings with compositions closer to natural bone apatites, including carbonates, trace elements are required. Selenium is an essential nutrient in biological tissues and, at the same time, it also presents antibacterial properties. A pioneering study on the fabrication of selenium-doped carbonated hydroxyapatite (iHA:Se) coatings by Pulsed Laser Deposition (PLD) is presented. Different proportions of selenium were incorporated to obtain the iHA:Se coatings. Their physicochemical characterization, performed by SEM/EDS, FTIR, FT-Raman, Interferometric Profilometry and XPS, revealed typical columnar growth of HA in globular aggregates and the efficient incorporation of selenium into the HA coatings by the, most probably, substitution of SeO(3)(2-) groups in the CO(3)(2-) sites. Biological evaluation illustrated the absence of cytotoxicity when an amount of 0.6 at.% of Se was added to the iHA:Se coatings and excellent proliferation of the MC3T3-E1 preosteoblasts. Antibacterial properties were also proved with the inhibition of P. aeruginosa and S. aureus from establishing bacterial biofilms.

An ice-templated, linearly aligned chitosan-alginate scaffold for neural tissue engineering

Abstract: Several strategies have been investigated to enhance axonal regeneration after spinal cord injury, however, the resulting growth can be random and disorganized. Bioengineered scaffolds provide a physical substrate for guidance of regenerating axons towards their targets, and can be produced by freeze casting. This technique involves the controlled directional solidification of an aqueous solution or suspension, resulting in a linearly aligned porous structure caused by ice templating. In this study, freeze casting was used to fabricate porous chitosan-alginate (C/A) scaffolds with longitudinally oriented channels. Chick dorsal root ganglia explants adhered to and extended neurites through the scaffold in parallel alignment with the channel direction. Surface adsorption of a polycation and laminin promoted significantly longer neurite growth than the uncoated scaffold (poly-L-ornithine + Laminin = 793.2 ± 187.2 μm; poly-L-lysine + Laminin = 768.7 ± 241.2 μm; uncoated scaffold = 22.52 ± 50.14 μm) (P < 0.001). The elastic modulus of the hydrated scaffold was determined to be 5.08 ± 0.61 kPa, comparable to reported spinal cord values. The present data suggested that this C/A scaffold is a promising candidate for use as a nerve guidance scaffold, because of its ability to support neuronal attachment and the linearly aligned growth of DRG neurites.

Enhanced osteoinductivity and osteoconductivity through hydroxyapatite coating of silk-based tissue-engineered ligament scaffold †

Abstract: Hybrid silk scaffolds combining knitted silk fibers and silk sponge have been recently developed for use as ligament-alone grafts. Incorporating an osteoinductive phase into the ends of a ligament scaffold may potentially generate an integrated "bone-ligament-bone" graft and improve graft osteointegration with host bone. To explore the possible application of hydroxyapatite (HA) coating in the fabrication of osteoinductive ends of silk-based scaffold, HA was coated on the hybrid silk scaffold and the effects to the bone-related cells were evaluated. HA could be coated in a uniform and controlled manner on the silk sponge, using an alternate soaking technology, with the amount deposited being dependent on the number of soaking cycles. HA coating also progressively reduced the hydrophobicity of silk surface (decreasing water contact angle from 87° to 42-76°, after 1-3 soaking cycles), making the HA-coated silk scaffold less favorable for initial cell attachments; but the attached cells showed viability and sustained proliferation on the HA-coated scaffold. As demonstrated by real-time polymerase chain reaction and alkaline phosphatase assay, the osteoinductivity of HA-coated silk scaffolds resulted in the osteogenic differentiation of bone marrow mesenchymal stem cells, and the osteoconductivity of HA-coated silk scaffolds supported osteoblasts growth and maintained the properties of mature osteoblasts. These properties of HA-coating demonstrated its possible application in fabricating osteoinductive ends of the silk-based ligament graft to potentially enhance graft-to-host bone integration.

Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering.

Abstract: In this study, a hierarchical nano/microfibrous chitosan/collagen scaffold that approximates structural and functional attributes of native extracellular matrix has been developed for applicability in skin tissue engineering. Scaffolds were produced by electrospinning of chitosan followed by imbibing of collagen solution, freeze-drying, and subsequent cross-linking of two polymers. Scanning electron microscopy showed formation of layered scaffolds with nano/microfibrous architechture. Physicochemical properties of scaffolds including tensile strength, swelling behavior, and biodegradability were found satisfactory for intended application. 3T3 fibroblasts and HaCaT keratinocytes showed good in vitro cellular response on scaffolds thereby indicating the matrices, cytocompatible nature. Scaffolds tested in an ex vivo human skin equivalent wound model, as a preliminary alternative to animal testing, showed keratinocyte migration and wound re-epithelization-a prerequisite for healing and regeneration. Taken together, the herein proposed chitosan/collagen scaffold, shows good potential for skin tissue engineering.

The roles of PI3K/Akt signaling pathway in regulating MC3T3-E1 preosteoblast proliferation and differentiation on SLA and SLActive titanium surfaces.

Abstract: Chemical modification to produce a hydrophilic microrough titanium (Ti) implant surface has been shown to increase osseointegration compared with microrough topography alone. This study aimed to investigate the roles of PI3K/Akt signaling pathway in regulating proliferation and differentiation of osteoblasts in response to surface microroughness and hydrophilicity. Ti disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT), a rough hydrophobic surface that was sand-blasted, large-grit, acid-etched (SLA), and an SLA surface with the same roughness that was chemically modified to possess high wettability/hydrophilicity (SLActive/modSLA). MC3T3-E1 cells were cultured on these substrates with or without LY294002, a PI3K inhibitor, and their behaviors, including cell viability (MTT colorimetric assay), alkaline phosphatase (ALP) activity, and osteogenic genes expression of osteopontin (OPN) and osteocalcin (OCN) were measured. Western blot was applied to detect the expression of PI3K/Akt signal pathway proteins. The results showed that a decrease in osteoblast proliferation associated with the Ti surfaces (SLActive > SLA > PT) correlated with an increase in activity of the osteogenic differentiation markers ALP. The peak of ALP activity appeared earlier at 7 days for the SLActive surfaces compared with the SLA and PT surfaces. Osteoblast proliferation, as well as the level of p-Akt, was significantly inhibited by LY294002 in all three Ti surfaces. The top value of ALP activity was increased with the inhibition of PI3K/Akt signaling pathway while the time of the peak appeared was not advanced. The expression levels of OPN and OCN were upregulated by the effect of surface roughness and hydrophilicity, which were further enhanced by LY294002. In conclusion, osteogenic responses to SLActive surface were moderately better than the SLA surface and protein expression studies indicated that PI3K/Akt signaling activation may be responsible for this increased osteogenic differentiation. Surface microroughness and hydrophilicity may affect osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation through PI3K/Akt signaling pathway.

Plasma treatment of electrospun PCL random nanofiber meshes (NFMs) for biological property improvement.

Abstract: In this article, the plasma surface modification effects on the chemical, mechanical, and biological properties of electrospun poly (e-caprolactone) (PCL) random nanofiber meshes (NFMs) were investigated by adjusting plasma chemistry, that is, using glow discharges of N(2) +H(2), NH(3) +O(2), and Ar+O(2) gas mixtures. The surface property changes of electrospun PCL NFMs after those plasma treatments were examined by water contact angle measurements and X-ray photoelectron spectroscopy. The experimental results showed that the plasma treatments introduced polar groups onto the surfaces and thus increased the surface hydrophilicity. From tensile test data, plasma treatment had limited effect on the mechanical properties of PCL random NFMs. The biological properties of the plasma-treated PCL NFMs were examined by cell proliferation assays using mouse osteoblast cells (MC3T3-E1). It was found that the plasma-treated PCL NFMs gave a higher proliferation rate and improved cell adhesion properties as compared with the untreated controls.

Bone integration capability of a series of strontium-containing hydroxyapatite coatings formed by micro-arc oxidation.

Abstract: Strontium-containing hydroxyapatites (Sr-HA) combine the desirable bone regenerative properties of hydroxyapatites (HA) with anabolic and anti-catabolic effects of strontium cations. In the present work, a series of Sr(y)HA [Sr(y)Ca(10-y)(PO4)6(OH)2; y = 0, 0.5, 1, 2] coatings on titanium are produced by micro-arc oxidation (MAO), and the effects of the in vivo osseointegration ability of the coatings are investigated by using a rabbit model. All samples are subjected to biomechanical, surface elemental, micro-CT and histological analysis after 4 and 12 weeks of healing. The obtained results show that the MAO-formed coatings exhibit a microporous network structure composed of Sr(y)HA/Sr(y)HA-Sr(x)Ca(1-x)TiO3/Sr(x)Ca(1-x)TiO3-TiO2 multilayers, in which the outer Sr(y)HA and intermediate Sr(y)HA-Sr(x)Ca(1-x)TiO3 layers have a nanocrystalline structure. All Sr-HA coated implants induce marked improvements in the behavior of bone formation, quantity and quality of bone tissue around the implants than the control HA implant and in particular, the 20%Sr-HA coating promotes early bone formation as identified by polyfluorochrome sequential labeling. The bone-to-implant contact is increased by 46% (p < 0.05) and the pull-out strength is increased by 103% over the HA group (p < 0.01). Extensive areas of mineralized tissue densely deposit on the 20%Sr-HA coating after biomechanical testing, and the greatest improvement of bone microarchitecture are observed around the 20%Sr-HA implant. The identified biological parameters successfully demonstrate the osteoconductivity of 20%Sr-HA surfaces, which results not only in an acceleration but also an improvement of bone-implant integration. The study demonstrates the immense potential of 20%Sr-HA coatings in dental and orthopedic applications.

Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium.

Abstract: Extracellular matrix (ECM) derived by tissue decellularization has applications as a tissue engineering scaffold and for support of cellular regeneration. Myocardial ECM from animals has been produced by whole-organ perfusion or immersion processes, but methods for preparation of human myocardial ECM for therapy and research have not been compared in detail, yet. We analyzed the impact of decellularization processes on human myocardial ECM, and tested its ability to serve as a scaffold for cell seeding. Sodium dodecyl sulfate (SDS)-based decellularization, but not treatments based on Triton X-100, deoxycholate or hypo/hypertonic incubations, removed cells satisfactorily, and incubation with fetal bovine serum (FBS) eliminated residual DNA. ECM architecture was best preserved by a protocol consisting of 2 h lysis, 6 h SDS, and 3 h FBS, but age and pathology of the donor tissue are highly important for producing reproducible, high-quality scaffolds. We also studied ECM repopulation with mesenchymal stem cells (CB-MSC), cardiomyocytes derived from induced pluripotent stem cells (iPS-CM), and na€ive neonatal mouse cardiomyocytes. Cells attached to the matrix and proliferated and displayed higher viability than in standard culture. We conclude that human cardiac ECM sheets may be suitable scaffold for cell-matrix interaction studies and as a biomaterial for tissue regeneration and engineering.

Rheological and mechanical properties of acellular and cell‐laden methacrylated gellan gum hydrogels

Abstract: Tissue engineered hydrogels hold great potential as nucleus pulposus substitutes (NP), as they promote intervertebral disc (IVD) regeneration and re-establish its original function. But, the key to their success in future clinical applications greatly depends on its ability to replicate the native 3D micro-environment and circumvent their limitation in terms of mechanical performance. In the present study, we investigated the rheological/mechanical properties of both ionic- (iGG-MA) and photo-crosslinked methacrylated gellan gum (phGG-MA) hydrogels. Steady shear analysis, injectability and confined compression stress-relaxation tests were carried out. The injectability of the reactive solutions employed for the preparation of iGG-MA and phGG-MA hydrogels was first studied, then the zero-strain compressive modulus and permeability of the acellular hydrogels were evaluated. In addition, human intervertebral disc (hIVD) cells encapsulated in both iGG-MA and phGG-MA hydrogels were cultured in vitro, and its mechanical properties also investigated under dynamic mechanical analysis at 37°C and pH 7.4. After 21 days of culturing, hIVD cells were alive (Calcein AM) and the E′ of ionic-crosslinked hydrogels and photo-crosslinked was higher than that observed for acellular hydrogels. Our study suggests that methacrylated gellan gum hydrogels present promising mechanical and biological performance as hIVD cells were producing extracellular matrix. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 3438–3446, 2013.

Development of polycaprolactone porous scaffolds by combining solvent casting, particulate leaching, and polymer leaching techniques for bone tissue engineering.

Abstract: Sodium chloride and polyethylene glycol (PEG) were used as water-soluble porogens for the formation of porous polycaprolactone (PCL) scaffolds. The main purpose was to prepare and evaluate in vitro efficacy of highly interconnected, three-dimensional, porous polymeric scaffolds, as obtained from the combined particulate and polymer leaching techniques. Microscopic analysis confirmed the high interconnectivity of the pores and relatively uniform pore size of 378–435 μm. The PCL scaffolds were further characterized for their density and pore characteristics, water absorption and flow behaviors, and mechanical properties and the potential for their use as bone scaffolding materials was evaluated in vitro using mouse calvaria-derived preosteoblastic cells (MC3T3-E1). Evidently, the use of PEG as the secondary porogen not only improved the interconnectivity of the pore structures but also resulted in the PCL scaffolds that exhibited much better support for the proliferation and differentiation of the cultured bone cells. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3379–3392, 2014.

Development of silver nanoparticle loaded antibacterial polymer mesh using plasma polymerization process.

Abstract: Plasma polymerized polyacrylic acid (PPAA) was deposited on a polymer substrate, namely polyethylene terephthalate (PET) mesh, for entrapment of silver nanoparticle (Ag-NP) in order to achieve antibacterial property to the material. Carboxylic groups of PPAA act as anchor as well as capping and stabilizing agents for Ag-NPs synthesized by chemical reduction method using NaBH4 as a reducing agent. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy (XPS), and water contact angle analysis were used to characterize the PPAA coatings. The Ag-NPs loaded polymer samples were characterized by UV–visible spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray, and XPS techniques. XPS analysis showed ∼1.0 at.% loading of Ag-NPs on to the PPAA-PET-mesh, which was composed of 79% zero-valent (Ag°) and 21% oxidized nano-Ag (Ag+). The plasma processed PET meshes samples were tested for antibacterial activity against two bacterial strains, namely Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative). Qualitative and quantitative tests showed that silver containing PPAA-PET meshes exhibit excellent antibacterial property against the tested bacteria with percent reduction of bacterial concentration >99%, compared to the untreated PET mesh. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Novel biomimetic thermosensitive β-tricalcium phosphate/chitosan-based hydrogels for bone tissue engineering.

Abstract: Among the less invasive surgical procedures for tissue engineering application, injectable in situ gelling systems have gained great attention. In this contest, this article is aimed to realize thermosensitive chitosan-based hydrogels, crosslinked with β-glycerophosphate and reinforced via physical interactions with β-tricalcium phosphate. The kinetics of sol-gel transition and the composite hydrogel properties were investigated by rheological analysis. The hydrogels were also characterized by Fourier transform infrared study, X-ray diffraction, scanning electron microscopy, transmission electron microscopy analysis, and thermal and biological studies. The hydrogels exhibit a gel-phase transition at body temperature, and a three-dimensional network with typical rheological properties of a strong gel. The presence of the inorganic phase, made up of nanocrystals, provides a structure with chemico-physical composition that mimics natural bone tissue, favoring cellular activity. These findings suggest the potential of the materials as promising candidates for hard tissue regeneration.