The influence of surface microtexture on osteogenesis was investigated in vitro by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as connective tissue progenitor cells (CTPs). Human bone marrow-derived CTPs were cultured for up to 60 days on smooth polydimethylsiloxane (PDMS) surfaces and on PDMS with post microtextures that were 10 µm in diameter and 6 µm in height, with 10 µm separation. DNA quantification revealed that the numbers of CTPs initially attached to both substrates were similar. However, cells on microtextured PDMS transitioned from lag phase after 4 days of culture, in contrast to 6 days for cells on smooth surfaces. By day 9 cells on the smooth surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation. In contrast, cells on the microtextured PDMS grew along the array of posts in an orthogonal manner. By days 30 and 60 cells grew and covered all surfaces with extracellular matrix. Western blot analysis revealed that the expression of integrin α5 was greater on the microtextured PDMS compared with smooth surfaces. Real time reverse transcription-polymerase chain reaction revealed that gene expression of alkaline phosphatase had decreased by days 30 and 60, compared with that on day 9, for both substrates. Gene expression of collagen I and osteocalcin was consistently greater on post microtextures relative to smooth surfaces at all time points.
Polydimethylsiloxane; Microfabrication; Soft lithography; Connective tissue progenitor cells; Adult stem cells
Nanogels based on poly(N-isopropylacrylamide) are attractive vehicles for prolonged duration local anesthesia because of their tunable size, number of functional groups, thermoresponsiveness, and their anionic charge. Nerve block durations of up to nine hours were achieved using acrylic acid-loaded nanogels loaded with bupivacaine. Increasing the anionic charge density of the nanogels or (for more highly acid-functionalized nanogels) decreasing the nanogel size facilitated longer duration anesthetic release. Small (<300 nm diameter) nanogels formed dense aggregates upon injection in vivo and induced only mild inflammatory responses, while large (>500 nm diameter) nanogels typically remained as liquid-like residues in vivo and induced more severe inflammatory reactions.
nanogels; poly(N-isopropylacrylamide); nerve block; local anesthesia; drug delivery
Injectable hydrogels derived from the extracellular matrix (ECM) of decellularized tissues have recently emerged as scaffolds for tissue engineering applications. Here, we introduce the potential for using a decellularized ECM-derived hydrogel for the improved delivery of heparin-binding growth factors. Immobilization of growth factors on a scaffold has been shown to increase their stability and activity. This can be done via chemical crosslinking, covalent bonding, or by incorporating natural or synthetic growth factor binding domains similar to those found in vivo in sulfated glycosaminoglycans (GAGs). Many decellularized ECM-derived hydrogels retain native sulfated GAGs and, therefore, these materials may provide an excellent delivery platform for heparin-binding growth factors. In this study, the sulfated GAG content of an ECM hydrogel derived from decellularized pericardial ECM was confirmed with FTIR and its ability to bind basic fibroblast growth factor (bFGF) was established. Delivery in the pericardial matrix hydrogel increased retention of bFGF both in vitro and in vivo in ischemic myocardium compared to delivery in collagen. In a rodent infarct model, intramyocardial injection of bFGF in pericardial matrix enhanced neovascularization by approximately 112% compared to delivery in collagen. Importantly, the newly formed vasculature was anastomosed with existing vasculature. Thus, the sulfated GAG content of the decellularized ECM hydrogel provides a platform for incorporation of heparin binding growth factors for prolonged retention and delivery.
We investigated the in vivo therapeutic efficacy of an intra-articular controlled release system consisting of biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating anti-inflammatory small interfering RNA (siRNA), together with branched poly(ethylenimine) (PEI) as a transfecting agent, in a rat model of painful temporomandibular joint (TMJ) inflammation. The in vivo effects of PLGA MP dose and siRNA-PEI polyplex delivery were examined via non-invasive meal pattern analysis and by quantifying the protein level of the siRNA target as well as of several downstream inflammatory cytokines. Controlled release of siRNA-PEI from PLGA MPs significantly reduced inflammation-induced changes in meal patterns compared to untreated rats with inflamed TMJs. These changes correlated to decreases in tissue-level protein expression of the siRNA target to 20–50% of the amount present in the corresponding control groups. Similar reductions were also observed in the expression of downstream inflammatory cytokines, e.g., interleukin-6 (IL-6), whose tissue levels in the siRNA-PEI PLGA MP groups were 50% of the values for the corresponding controls. This intra-articular sustained release system has significant implications for the treatment of severe TMJ pain, and also has the potential to be readily adapted and applied to mitigate painful, chronic inflammation in a variety of conditions.
Controlled drug release; drug delivery; microsphere; inflammation; immunomodulation
Fibrin and alginate hydrogels have been widely used to support chondrogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs) for articular cartilage and fibrocartilage tissue engineering, with distinct advantages and disadvantages to each material. Attempting to produce a gel scaffold exhibiting beneficial characteristics of both materials, we fabricated fibrin/alginate blended hydrogels at various blend ratios and evaluated the gel morphology, mechanical properties and their support for BM-MSC chondrogenesis. Results show that when the fibrin/alginate ratio decreased, the fibrin architecture transitioned from uniform to interconnected fibrous and finally to disconnected islands against an alginate background, with opposing trends in the alginate architecture. Fibrin maintained gel extensibility and promoted cell proliferation, while alginate improved the gel biostability and better supported glycosaminoglycan and collagen II production and chondrogenic gene expression. Blended gels had physical and biological characteristics intermediate between fibrin and alginate. Of the blends examined, FA 40:8 (40 mg/mL fibrinogen blended with 8 mg/mL alginate) was found to be the most appropriate group for future studies on tension-driven BM-MSC fibrochondrogenesis. As BM-MSC differentiation appeared to vary between fibrin and alginate regions of blended scaffolds, this study also highlighted the potential to develop spatially heterogeneous tissues through manipulating the heterogeneity of scaffold composition.
Chondrogenesis; mesenchymal stem cells; fibrin; alginate; hydrogel
The spinal cord (SC) and dorsal root ganglion (DRG) are target implantation regions for neural prosthetics, but the tissue-electrode interface in these regions is not well-studied. To improve our understanding of these locations, we characterized the tissue reactions around implanted electrodes. L1, an adhesion molecule shown to maintain neuronal density and reduce gliosis in brain tissue, was then evaluated in SC and DRG implants. Following L1 immobilization onto neural electrodes, the bioactivities of the coatings were verified in vitro using neuron, astrocyte and microglia cultures. Non-modified and L1-coated electrodes were implanted into adult rats for 1 or 4 weeks. Hematoxylin and eosin staining along with cell-type specific antibodies were used to characterize the tissue response. In the SC and DRG, cells aggregated at the electrode-tissue interface. Microglia staining was more intense around the implant site and decreased with distance from the interface. Neurofilament staining in both locations was decreased or absent around the implant when compared to surrounding tissue. With L1, neurofilament staining was significantly increased while neuronal cell death decreased. Our results indicate that L1-modified electrodes may result in an improved chronic neural interface and will be evaluated in recording and stimulation studies.
electrode; interface; biocompatibility; neural prosthesis; surface modification
Few therapeutic options exist for meniscus repair after injury. Local delivery of growth factors may stimulate repair and create a favorable environment for engineered replacement materials. In this study, we assessed the effect of basic fibroblast growth factor (bFGF) (a pro-mitotic agent) and transforming growth factor beta 3 (TGF-β3) (a pro-matrix formation agent) on meniscus repair and the integration/maturation of electrospun poly(ε-caprolactone) (PCL) scaffolds for meniscus tissue engineering. Circular meniscus repair constructs were formed and refilled with either native tissue or scaffolds. Repair constructs were cultured in serum-containing media for 4 and 8 weeks with various growth factor formulations, and assessed for mechanical strength, biochemical content, and histological appearance. Results showed that either short-term delivery of bFGF or sustained delivery of TGF-β3 increased integration strength for both juvenile and adult bovine tissue, with similar findings for engineered materials. While TGF-β3 increased proteoglycan content in the explants, bFGF did not increase DNA content after 8 weeks. This work suggests that in vivo delivery of bFGF or TGF-β3 may stimulate meniscus repair, but that the time course of delivery will strongly influence success. Further, this study demonstrates that electrospun scaffolds are a promising material for meniscus tissue engineering, achieving comparable or superior integration compared to native tissue.
Meniscus; Tissue Engineering; Growth Factors; Scaffolds
Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal-epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment, and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36%–54% increase on average in initial cell attachment, which corresponded to an over 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially in regards to FA distribution, in vitro.
cornea; epithelium; focal adhesion; topography; silk
Injectable biomaterials alone may alter local tissue responses, including inflammatory cascades and matrix production (e.g., stimulatory dermal fillers are used as volumizing agents that induce collagen production). To expand upon the available material compositions and timing of presentation, a tunable hyaluronic acid (HA) and poly(lactide-co-glycolide) (PLGA) microsphere composite system was formulated and assessed in subcutaneous and cardiac tissues. HA functionalized with hydroxyethyl methacrylate (HeMA) was used as a precursor to injectable and degradable hydrogels that carry PLGA microspheres (~50 m diameter) to tissues, where the HA hydrogel degradation (~20 or 70 days) and quantity of PLGA microspheres (0–300 mg/ml) are readily varied. When implanted subcutaneously, faster hydrogel degradation and more microspheres (e.g., 75mg/mL) generally induced more rapid tissue and cellular interactions and a greater macrophage response. In cardiac applications, tissue bulking may be useful to alter stress profiles and to stabilize the tissue after infarction, limiting left ventricular (LV) remodeling. When fast degrading HeMA-HA hydrogels containing 75 mg/mL microspheres were injected into infarcted tissue in sheep, LV dilation was limited and the thickness of the myocardial wall and the presence of vessels in the apical infarct region were increased ~35% and ~60%, respectively, compared to empty hydrogels. Both groups decreased volume changes and infarct areas at 8 weeks, compared to untreated controls. This work illustrates the importance of material design in expanding the application of tissue bulking composites to a range of biomedical applications.
hyaluronic acid; hydrogel; microspheres; remodeling; myocardial infarction
Human embryonic stem cells (hESCs) are exciting for regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there has been no report on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESCs were cultured to form embryoid bodies (EBs), and the MSCs were then migrated out of the EBs. hESCd-MSCs had surface markers characteristic of MSCs, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSCs were encapsulated in alginate at a density of 1 million cells/mL, with an average microbead size of 207 µm. CPC contained mannitol porogen to create a porosity of 64% and macropores with size of 218 µm, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSCs encapsulated in microbeads in CPC had good viability from 1 to 21 d. ALP gene expression at 21 d was 25-fold that at 1 d. Osteocalcin (OC) at 21 d was two orders of magnitude of that at 1 d. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21 d was 5-fold that at 1 d. Mineral synthesis by the encapsulated hESCd-MSCs at 21 d was 7-fold that at 1 d. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSCs were encapsulated in alginate microbeads in macroporous CPC showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.
Human embryonic stem cells; calcium phosphate cement; macroporosity; injectable scaffold; osteogenic differentiation; bone tissue engineering
Traumatic brain injuries have recently been put under the spotlight as one of the most important causes of accidental brain dysfunctions. Significant experimental and modeling efforts are thus ongoing to study the associated biological, mechanical and physical mechanisms. In the field of cell mechanics, progresses are also being made at the experimental and modeling levels to better characterize many of the cell functions such as differentiation, growth, migration and death, among others. The work presented here aims at bridging both efforts by proposing a continuum model of neuronal cell submitted to blast loading. In this approach, cytoplasm, nucleus and membrane (plus cortex) are differentiated in a representative cell geometry, and different material constitutive models are adequately chosen for each one. The material parameters are calibrated against published experimental work of cell nanoindentation at multiple rates. The final cell model is ultimately subjected to blast loading within a complete fluid-structure interaction computational framework. The results are compared to the nanoindentation simulation and the specific effects of the blast wave on the pressure and shear levels at the interfaces are identified. As a conclusion, the presented model successfully captures some of the intrinsic intracellular phenomena occurring during its deformation under blast loading and potentially leading to cell damage. It suggests more particularly the localization of damage at the nucleus membrane similarly to what has already been observed at the overall cell membrane. This degree of damage is additionally predicted to be worsened by a longer blast positive phase duration. As a conclusion, the proposed model ultimately provides a new three dimensional computational tool to evaluate intracellular damage during blast loading.
continuum model; neuron; blast; cell damage; traumatic brain injury
Design of antimicrobial polymers for enhancing healthcare issues and minimizing environmental problems is an important endeavor with both fundamental and practical implications. Quaternary ammonium silane-functionalized methacrylate (QAMS) represents an example of antimicrobial macromonomers synthesized by a sol-gel chemical route; these compounds possess flexible Si-O-Si bonds. In present work, a partially-hydrolyzed QAMS copolymerized with bis-GMA is introduced. This methacrylate resin was shown to possess desirable mechanical properties with both a high degree of conversion and minimal polymerization shrinkage. Kill-on-contact microbiocidal activities of this resin were demonstrated using single-species biofilms of Streptococcus mutans (ATCC 36558), Actinomyces naeslundii (ATCC 12104) and Candida albicans (ATCC 90028). Improved mechanical properties after hydration provided the proof-of-concept that QAMS-incorporated resin exhibits self-repair potential via water-induced condensation of organic modified silicate (ormosil) phases within the polymerized resin matrix.
Quaternary ammonium; Organic modified silicate; Antimicrobial; Sol-gel technique; Self-repair
Although various cell encapsulation materials are available commercially for a wide range of potential therapeutic cells, their combined clinical impact remains inconsistent. Synthetic materials such as poly(ethylene glycol) (PEG) hydrogels are mechanically robust and have been extensively explored but lack natural biofunctionality. Naturally derived materials including collagen, fibrin and alginate-chitosan are often labile and mechanically weak. In this paper we report the development of a hybrid biomatrix based on the thiol-ene reaction of PEG diacrylate (PEGdA) and cysteine/PEG-modified gelatin (gel-PEG-Cys). We hypothesized that covalent crosslinking decreases gelatin dissolution thus increasing gelatin resident time within the matrix and the duration of its biofunctionality; at the same time the relative ratio of PEGdA to gel-PEG-Cys in the matrix formulation directly affects hydrogel bulk and local microenvironment properties. Bulk viscoelastic properties were highly dependent on PEGdA concentration and total water content, while gel-PEG-Cys concentration was more critical to swelling profiles. Microviscoelastic properties were related to polymer concentration. The covalently crosslinked gel-PEG-Cys with PEGdA decreased gelatin dissolution out of the matrix and collagenase-mediated degradation. Fibroblasts and keratinocyte increased adhesion density and formed intercellular connections on stiffer hydrogel surfaces, while cells exhibited more cytoplasmic spreading and proliferation when entrapped within softer hydrogels. Hence, this material system contains multiparametric factors that can easily be controlled to modulate the chemical, physical and biological properties of the biomatrix for soft tissue scaffolding and cell presentation to reconstruct lost tissue architecture and physical functionality.
Bioactivity; Cell encapsulation; Biomatrix; Gelatin; Poly(ethylene glycol)
The enamel of human teeth is generally regarded as a brittle material with low fracture toughness. Consequently, the contributions of this tissue in resisting tooth fracture and the importance of its complex microstructure have been largely overlooked. In this study an experimental evaluation of the crack growth resistance of human enamel was conducted to characterize the role of rod (i.e. prism) orientation and degree of decussation on the fracture behavior of this tissue. Incremental crack growth was achieved in-plane, with the rods in directions longitudinal or transverse to their axes. Results showed that the fracture resistance of enamel is both inhomogeneous and spatially anisotropic. Cracks extending transverse to the rods in the outer enamel undergo a lower rise in toughness with extension, and achieve significantly lower fracture resistance than in the longitudinal direction. Though cracks initiating at the surface of teeth may begin extension towards the dentin–enamel junction, they are deflected by the decussated rods and continue growth about the tooth’s periphery, transverse to the rods in the outer enamel. This process facilitates dissipation of fracture energy and averts cracks from extending towards the dentin and vital pulp.
Crack growth; Enamel; Fracture toughness; Strength
β-Tricalcium phosphate (β-TCP) is an attractive ceramic for bone tissue repair because of its similar composition to bone mineral and its osteoconductivity. However, compared with other ceramics β-TCP has a rapid and uncontrolled rate of degradation. In the current study β-TCP granules were mineral coated with the aim of influencing the dissolution rate of β-TCP, and also to use the coating as a carrier for controlled release of the growth factors recombinant human vascular endothelial growth factor (rhVEGF), modular VEGF peptide (mVEGF), and modular bone morphogenetic protein 2 peptide (mBMP2). The biomineral coatings were formed by heterogeneous nucleation in aqueous solution using simulated body fluid solutions with varying concentrations of bicarbonate (HCO3). Our results demonstrate that we could coat β-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the β-TCP granules. As the carbonate (CO32−) content in the coating was increased the dissolution rate of the coated β-TCP also increased, but remained slower than the dissolution of uncoated β-TCP. In addition, we showed sustained release of multiple growth factors, with release kinetics that were controllable by varying the identity of the growth factor or the CO32− con-tent in the mineral coating. Released rhVEGF induced human umbilical vein endothelial cell (HUVEC) proliferation, and mVEGF enhanced migration of mouse embryonic endothelial cells in a scratch wound healing assay, indicating that each released growth factor was biologically active.
β-TCP; Bone morphogenetic protein 2; Vascular endothelial growth factor; Bone tissue engineering; Hydroxyapatite
Engineered cartilage based on adult mesenchymal stem cells (MSCs) is an alluring goal for the repair of articular defects. However, efforts to date have failed to generate constructs with sufficient mechanical properties to function in the demanding environment of the joint. Our findings with a novel photocrosslinked hyaluronic acid (HA) hydrogel suggest that stiff gels (high HA concentration, 5% w/vol) foster chondrogenic differentiation and matrix production, but limit overall functional maturation due to the inability of formed matrix to diffuse away from the point of production and form a contiguous network. In the current study, we hypothesized that increasing the MSC seeding density would decrease the required diffusional distance, and so expedite the development of functional properties. To test this hypothesis, bovine MSCs were encapsulated at seeding densities of either 20 or 60 million cells per mL in 1%, 3%, and 5% (w/vol) hyaluronic acid (HA) hydrogels. Counter our hypothesis, higher concentration HA gels (3% and 5%) did not develop more rapidly with increased MSC seeding density. However, the biomechanical properties of low concentration (1%) HA constructs increased markedly (nearly 3-fold with a 3-fold increase in seeding density). To ensure that optimal nutrient access was delivered, we next cultured these constructs under dynamic culture conditions (orbital shaking) for 9 weeks. Under these conditions, 1% HA seeded at 60 million MSCs per mL reached a compressive modulus in excess of 1 MPa (compared to 0.3-0.4MPa for free swelling constructs). This is the highest level we have reported to date in this HA hydrogel system, and represents a significant advance towards functional stem cell-based tissue engineered cartilage.
cartilage; hydrogel; tissue engineering; hyaluronic acid / hyaluronan; mesenchymal stem cell
New magnetic-based core-shell particles (MBCSP) were developed to target skin cancer cells while delivering chemotherapeutic drugs in a controlled fashion. MBCSP consist of a thermo-responsive shell of poly(N-isopropylacrylamide-acrylamide-allylamine) and a core of poly(lactic-co-glycolic acid) (PLGA) embedded with magnetite nanoparticles. To target melanoma cancer cells, MBCSP were conjugated with Gly-Arg-Gly-Asp-Ser (GRGDS) peptides that specifically bind to the α5β3+ receptor of melanoma cell. MBCSP consist of unique multifunctional and controlled drug delivery characteristics. Specially, they can provide dual drug release mechanisms (a sustained release of drugs through degradation of PLGA core and a controlled release in response to changes in temperature via thermo-responsive polymer shell), and dual targeting mechanisms (magnetic localization and receptor-mediated targeting). Results from in vitro studies indicate that GRGDS-conjugated MBCSP has an average diameter of 296 nm and exhibit no cytotoxicity towards human dermal fibroblasts up to 500 μg ml−1. Further, a sustained release of curcumin from the core and a temperature-dependent release of doxorubicin from the shell of MBCSP were observed. The particles also produced a dark contrast signal in magnetic resonance imaging. Finally, the particles were accumulated at the tumor site in a B16F10 melanoma orthotopic mouse model, especially in presence of a magnet. Results indicate great potential of MBCSP as a platform technology to target, treat, and monitor melanoma for targeted drug delivery to reduce side effects of chemotherapeutic reagents.
Biodegradable polymers; Magnetite nanoparticles; Thermo-responsive polymers; Targeted drug delivery; Melanoma
Bioresorbable scaffolds with mechanical properties suitable for bone tissue engineering were fabricated from polycaprolactone (PCL) and hydroxyapatite (HA) by selective laser sintering (SLS) and modeled by finite element analysis (FEA). Both solid gage parts and scaffolds having 1-D, 2-D and 3-D orthogonal, periodic porous architectures were made with 0, 10, 20 and 30% HA by volume. PCL:HA scaffolds manufactured by SLS had nearly full density (99%) in the designed solid regions and had excellent geometric and dimensional control. Through optimization of the SLS process, the compressive moduli for our solid gage parts and scaffolds are the highest reported in the literature for additive manufacturing. The compressive moduli of solid gage parts were 299.3, 311.2, 415.5 and 498.3 MPa for PCL:HA loading at 100:0, 90:10, 80:20 and 70:30 respectively. The compressive effective stiffness tended to increase as the loading of HA was increased and the designed porosity was lowered. In the case of the most 3-D porous scaffold, the compressive modulus more than doubled from 14.9 MPa to 36.2 MPa when changing the material from 100:0 to 70:30 PCL:HA. A micromechanical finite element analysis (FEA) model was developed to investigate the reinforcement effect of HA loading on the compressive modulus of the bulk material. Using a first-principles based approach, the random distribution of HA particles in a solidified PCL matrix was modeled for any loading of HA to predict the bulk mechanical properties of the composites. The bulk mechanical properties were also used for FEA of the scaffold geometries. Results of the FEA were found to be in good agreement with experimental mechanical testing. The development of patient and site-specific composite tissue engineering constructs with tailored properties can be seen as a direct extension of this work on computational design, a priori modeling of mechanical properties and direct digital manufacturing.
tissue engineering; scaffolds; polycaprolactone; hydroxyapatite; selective laser sintering; mechanical properties
Infection in primary total joint prostheses is estimated to occur in up to 3% of all surgeries. As a measure to improve the antimicrobial properties of implant materials, silver (Ag) was incorporated into plasma sprayed hydroxyapatite (HA) coatings. To offset potential cytotoxic effects of Ag in the coatings, strontium (Sr) was also added as a binary dopant. HA powder were doped with 2.0 wt% Ag2O, 1.0 wt% SrO and the powder was then heat treated at 800° C. Titanium substrates were coated using a 30 kW plasma spray system equipped with a supersonic nozzle. X-ray diffraction (XRD) confirmed the phase purity and high crystallinity of the coatings. Samples were evaluated for mechanical stability by adhesive bond strength testing. Results show that the addition of dopants did not affect the overall bond strength of the coatings. The antibacterial efficacies of the coatings were tested against Pseudomonas aeruginosa. Samples that contained the Ag2O dopant were found to be highly effective against the bacterial colonization. In vitro cell-material interactions using human fetal osteoblast (hFOB) cells were characterized by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability, field emission scanning electron microscopy (FESEM) for cell morphology and confocal imaging for the important differentiation marker alkaline phosphatase (ALP). Our results showed evidence of cytotoxic effects in the Ag-HA coatings, characterized by poor cellular morphology and cell death and nearly complete impediment of functional ALP activity. The addition of SrO to Ag-HA coatings was able to effectively offset these negative effects and improve the performance when compared to pure HA coated samples.
hydroxyapatite; coating; antibacterial; silver; strontium
The extracellular matrix (ECM) of mammalian tissues has been isolated, decellularized and utilized as a scaffold to facilitate the repair and reconstruction of numerous tissues. Recent studies have suggested that superior function and complex tissue formation occurred when ECM scaffolds were derived from site-specific homologous tissues compared with heterologous tissues. The objectives of the present study were to apply a stringent decellularization process to demineralized bone matrix (DBM), prepared from bovine bone, and to characterize the structure and composition of the resulting ECM materials and DBM itself. Additionally, we sought to produce a soluble form of DBM and ECM which could be induced to form a hydrogel. Current clinical delivery of DBM particles for treatment of bone defects requires incorporation of the particles within a carrier liquid. Differences in osteogenic activity, inflammation and nephrotoxicity have been reported with various carrier liquids. The use of hydrogel forms of DBM or ECM may reduce the need for carrier liquids. DBM and ECM hydrogels exhibited sigmoidal gelation kinetics consistent with a nucleation and growth mechanism, with ECM hydrogels characterized by lower storage moduli than the DBM hydrogels. Enhanced proliferation of mouse primary calvarial cells was achieved on ECM hydrogels, compared with collagen type I and DBM hydrogels. These results show that DBM and ECM hydrogels have distinct structural, mechanical and biological properties and have the potential for clinical delivery without the need for carrier liquids.
Extracellular matrix; Demineralized bone matrix; Hydrogel; Bone graft; Decellularization
The interactions of cancer cells within a solid mass with the surrounding reactive stroma are critical for growth and progression. The surrounding vasculature is recruited into the periphery of the growing tumour to supply cancer cells with nutrients and O2. This study focuses on developing a novel three-dimensional (3-D) in vitro biomimetic colorectal cancer model using colorectal cancer cells and connective tissue cells. The 3-D model comprises a dense artificial cancer mass, created by partial plastic compression of collagen type I containing HT29 colorectal cancer cells, nested in a non-dense collagen type I gel populated by fibroblasts and/or endothelial cells. HT29 cells within the dense mass proliferate slower than when cultured in a two-dimensional system. These cells form tumour spheroids which invade the surrounding matrix, away from the hypoxic conditions in the core of the construct, measured using real time O2 probes. This model is also characterized by the release of vascular endothelial growth factor (VEGF) by HT29 cells, mainly at the invading edge of the artificial cancer mass. This characterization is fundamental in establishing a reproducible, complex model that could be used to advance our understanding of cancer pathology and will facilitate therapeutic drug testing.
Collagen I; Hypoxia; Plastic compression; Three-dimensional culture; Tumour microenvironment; Tumour invasion
Currently, younger, more active patients are being offered total joint replacement (TJR) for end-stage arthritic disorders. Despite improved durability of TJRs, particle-associated wear of the bearing surfaces continues to be associated with particulate debris, which can activate monocyte/macrophages. Activated macrophages then produce pro-inflammatory factors and cytokines that induce an inflammatory reaction that activates osteoclasts leading to bone breakdown and aseptic loosening. We hypothesized that activated macrophages in tissues harvested from revised joint replacements predominantly express an M1 pro-inflammatory phenotype due to wear-particle-associated cell activation, rather than an M2 antiinflammatory phenotype. We further questioned whether it is possible to convert uncommitted monocyte/macrophages to an M2 phenotype by the addition of interleukin-4 (IL-4), or whether it is necessary to first pass through an M1 intermediate stage. Retrieved periprosthetic tissues demonstrated increased M1/M2 macrophage ratios compared to non-operated osteoarthritic synovial tissues, using immunohistochemical staining and Western blotting. Uncommitted monocyte/macrophages with/without poly-methyl-methacrylate particles were transformed to an M2 phenotype by IL-4 more efficiently when the cells were first passed through an M1 phenotype by exposure to endotoxin. Wear particles induce a pro-inflammatory microenvironment that facilitates osteolysis; these events may potentially be modulated favorably by exposure to IL-4.
M1 M2 macrophages; Osteolysis; Total joint replacement; PMMA; IL-4
Load-bearing tissues owe their mechanical strength to their highly-anisotropic collagenous structure. To date, attempts to engineer mechanically strong connective tissue have failed mainly due to the lack of the ability to reproduce native collagen organization in constructs synthesized by cultured cells in vitro. The ability to influence the direction of the self-assembling collagen molecules and produce highly anisotropic structures has applications ranging from de novo engineering of complex tissues to the production of organized scaffolds for cell culture contact guidance. In this investigation we have used the simple technique of spin coating to produce highly-aligned arrays of collagen fibrils. By a simple modification of the method we have also successfully produced orthogonal collagen lamellae. Alternating collagen lamellae are frequently seen in load-bearing tissues such as cornea, annulus fibrosus, and cortical bone. Culturing of corneal fibroblasts onto aligned collagen shows that the cells adopt the organization of the fibrils. In this investigation, we observed the reversal of fibrillar growth direction or “hook” formation similar to those seen previously in a microfluidic shear-flow chamber. Although the results of this investigation clearly show that it is possible to produce small areas (O) 1 cm2 of collagen fibrils with enough alignment to guide fibroblasts, there is evidence that thin film instabilities are likely to be a significant barrier to producing organized collagen fibrils over larger areas. Successful application of this method to produce highly-controlled and organized collagenous structures will require the development of techniques to control thin film instability and will be the subject of the future work.
Collagen; Corneal Tissue Engineering; Extracellular Matrix; Shear Stress
The present studies were designed to evaluate the adjuvant activity of polyanhydride microparticles prepared in the absence of additional stabilizers, excipients, or immune modulators. Microparticles composed of varying ratios of either 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) or 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH were added to in vitro cultures of bone marrow-derived dendritic cells (DCs). Microparticles were efficiently and rapidly phagocytosed by DCs in the absence of opsonization and without centrifugation or agitation. Within 2 h, internalized particles were rapidly localized to an acidic, phagolysosomal compartment. By 48 h, only a minor reduction in microparticle size was observed in the phagolysosomal compartment, indicating minimal particle erosion consistent with being localized within an intracellular microenvironment favoring particle stability. Polyanhydride microparticles increased DC surface expression of MHC II, the co-stimulatory molecules CD86 and CD40, and the C-type lectin CIRE (murine DC-SIGN; CD209). In addition, microparticle stimulation of DCs also enhanced secretion of the cytokines IL-12p40 and IL-6, a phenomenon found to be dependent on polymer chemistry. DCs cultured with polyanhydride microparticles and ovalbumin induced polymer chemistry-dependent antigen-specific proliferation of both CD4+ OT-II and CD8+ OT-I T cells. These data indicate that polyanhydride particles can be tailored to take advantage of the potential plasticity of the immune response, resulting in the ability to induce immune protection against many types of pathogens.
Polyanhydrides; Dendritic cells; Adjuvants; Vaccine delivery; Microparticles
Controlled scaffold degradation is a critical design criterion for the clinical success of tissue engineered constructs. Here, we exploited a biomimetic poly(ethylene glycol) diacrylate (PEGDA) hydrogel system immobilized with tethered YRGDS as the cell adhesion ligand and with either single (SSite) or multiple (MSite) collagenase-sensitive domains between crosslinks, to systematically study the effect of proteolytic cleavage site presentation on hydrogel degradation rate and 3D fibroblast invasion in vitro. Through the incorporation of multiple collagenase-sensitive domains between crosslinks, hydrogel degradation rate was controlled and enhanced independent of alterations in compressive modulus. As compared to SSite hydrogels, MSite hydrogels resulted in increased 3D fibroblast invasion in vitro which occurred over a wider range of compressive modulus. Furthermore, encapsulated soluble acidic fibroblast growth factor (FGF-1), a potent mitogen during processes such as vascularization and wound healing, was incorporated into SSite and MSite PEGDA scaffolds to determine its in vitro potential on fibroblast cell invasion. Hydrogels containing soluble FGF-1 significantly enhanced 3D fibroblast invasion in a dose-dependent manner within the different types of PEG matrices investigated over a period of 15 days. The methodology presented provides flexibility in designing PEG scaffolds with desired mechanical properties, but with increased susceptibility to proteolytically-mediated degradation. These results indicate that effective tuning of initial matrix stiffness and hydrogel degradation kinetics plays a critical role in effectively designing PEG scaffolds that promote controlled 3D cellular behavior and in situ tissue regeneration.
Biodegradation; Biomimetic Material; Free-radical; Cross-linking; Fibroblast growth factor; fibroblasts