Effect of Fibrillization pH on Gelation Viscoelasticity and Properties of Biofabricated Dense Collagen Matrices via Gel Aspiration-Ejection.

Reconstituted hydrogels based on the self-assembly of acid-solubilized collagen molecules have been extensively used as in vitro models and precursors in biofabrication processes. This study investigated the effect of fibrillization pH-ranging from 4 to 11-on real-time rheological property changes during the gelation of collagen hydrogels and its interplay with the properties of subsequently biofabricated dense collagen matrices generated via automated gel aspiration-ejection (GAE). A contactless, nondestructive technique was used to characterize the temporal progression in shear storage modulus (G', or stiffness) during collagen gelation. There was a relative increase in G' of the hydrogels from 36 to 900 Pa with an increase in gelation pH. Automated GAE, which simultaneously imparts collagen fibrillar compaction and alignment, was then applied to these precursor collagen hydrogels to biofabricate native extracellular matrix-like densified gels. In line with viscoelastic properties, only hydrogels fibrillized in the 6.5 < pH ≤ 10 range could be densified via GAE. There was an increase in both fibrillar density and alignment in the GAE-derived matrices with an increase in gelation pH. These factors, combined with a higher G' in the alkaline precursor hydrogels, led to a significant increase in the micro-compressive modulus of GAE-densified gels of pH 9 and 10. Furthermore, NIH/3T3 fibroblast-seeded GAE-derived matrices densified from gels fibrillized in the pH range of 7 to 10 exhibited low cell mortality with >80% viability. It is anticipated that the results of this study can be potentially applicable to other hydrogel systems, as well as biofabrication techniques involving needles or nozzles, such as injection and bioprinting.

Multiscale structural evolution of citrate-triggered intrafibrillar and interfibrillar mineralization in dense collagen gels.

The mineralized extracellular matrix of bone is an organic-inorganic nanocomposite consisting primarily of carbonated hydroxyapatite, fibrous type I collagen, noncollagenous proteins, proteoglycans, and diverse biomolecules such as pyrophosphate and citrate. While much is now known about the mineralization-regulating role of pyrophosphate, less is known about the function of citrate. In order to assess the effect of negatively charged citrate on collagen mineralization, citrate-functionalized, bone osteoid-mimicking dense collagen gels were exposed to simulated body fluid for up to 7 days to examine the multiscale evolution of intra- and interfibrillar collagen mineralization. Here, we show by increases in methylene blue staining that the net negative charge of collagen can be substantially augmented through citrate functionalization. Structural and compositional analyses by transmission and scanning electron microscopy (including X-ray microanalysis and electron diffraction), and atomic force microscopy, all demonstrated that citrate-functionalized collagen fibrils underwent extensive intrafibrillar mineralization within 12 h in simulated body fluid. Time-resolved, high-resolution transmission electron microscopy confirmed the temporal evolution of intrafibrillar mineralization of single collagen fibrils. Longer exposure to simulated body fluid resulted in additional interfibrillar mineralization, all through an amorphous-to-crystalline transformation towards apatite (assessed by X-ray diffraction and attenuated total reflection-Fourier-transform infrared spectroscopy). Calcium deposition assays indicated a citrate concentration-dependent temporal increase in mineralization, and micro-computed tomography confirmed that >80 vol% of the collagen in the gels was mineralized by day 7. In conclusion, citrate effectively induces mesoscale intra- and interfibrillar collagen mineralization, a finding that advances our understanding of the role of citrate in mineralized tissues.

Mineralization of Bone Extracellular Matrix-like Scaffolds Fabricated as Silk Sericin-Functionalized Dense Collagen-Fibrin Hybrid Hydrogels.

The design of hydrogels that combine both the biochemical cues needed to direct seeded cellular functions and mineralization to provide the structural and mechanical properties approaching those of mineralized native bone extracellular matrix (ECM) represents a significant challenge in bone tissue engineering. While fibrous hydrogels constituting of collagen or fibrin (and their hybrids) can be considered as scaffolds that mimic to some degree native bone ECM, their insufficient mechanical properties limit their application. In the present study, an automated gel aspiration-ejection (automated GAE) method was used to generate collagen-fibrin hybrid gel scaffolds with micro-architectures and mechanical properties approaching those of native bone ECM. Moreover, the functionalization of these hybrid scaffolds with negatively charged silk sericin accelerated their mineralization under acellular conditions in simulated body fluid and modulated the proliferation and osteoblastic differentiation of seeded MC3T3-E1 pre-osteoblastic cells. In the latter case, alkaline phosphatase activity measurements indicated that the hybrid gel scaffolds with seeded cells showed accelerated osteoblastic differentiation, which in turn led to increased matrix mineralization. In summary, the design of dense collagen-fibrin hybrid gels through an automated GAE process can provide a route to tailoring specific biochemical and mechanical properties to different types of bone ECM-like scaffolds, and can provide a model to better understand cell-matrix interactions in vitro for bioengineering purposes.

Factor XIIIA transglutaminase expression and secretion by osteoblasts is regulated by extracellular matrix collagen and the MAP kinase signaling pathway.

Osteoblast differentiation is regulated by the presence of collagen type I (COL I) extracellular matrix (ECM). We have recently demonstrated that Factor XIIIA (FXIIIA) transglutaminase (TG) is required by osteoblasts for COL I secretion and extracellular deposition, and thus also for osteoblast differentiation. In this study we have further investigated the link between COL I and FXIIIA, and demonstrate that COL I matrix increases FXIIIA levels in osteoblast cultures and that FXIIIA is found as cellular (cFXIIIA) and extacellular matrix (ecmFXIIIA) forms. FXIIIA mRNA, protein expression, cellular localization and secretion were enhanced by ascorbic acid (AA) treatment and blocked by dihydroxyproline (DHP) which inhibits COL I externalization. FXIIIA mRNA was regulated by the MAP kinase pathway. Secretion of ecmFXIIIA, and its enzymatic activity in conditioned medium, were also decreased in osteoblasts treated with the lysyl oxidase inhibitor ß-aminopropionitrile, which resulted in a loosely packed COL I matrix. Osteoblasts secrete a latent, inactive dimeric ecmFXIIIA form which is activated upon binding to the matrix. Monodansyl cadaverine labeling of TG substrates in the cultures revealed that incorporation of the label occurred at sites where fibronectin co-localized with COL I, indicating that ecmFXIIIA secretion could function to stabilize newly deposited matrix. Our results suggest that FXIIIA is an integral part of the COL I deposition machinery, and also that it is part of the ECM-feedback loop, both of which regulate matrix deposition and osteoblast differentiation.

Mechanical activation drives tenogenic differentiation of human mesenchymal stem cells in aligned dense collagen hydrogels.

Tendons are force transmitting mechanosensitive tissues predominantly comprised of highly aligned collagen type I fibres. In this study, the recently introduced gel aspiration-ejection method was used to rapidly fabricate aligned dense collagen (ADC) hydrogel scaffolds. ADCs provide a biomimetic environment compared to traditional collagen hydrogels that are mechanically unstable and comprised of randomly oriented fibrils. The ADC scaffolds were shown to be anisotropic with comparable stiffness to immature tendons. Furthermore, the application of static and cyclic uniaxial loading, short-term (48 h) and high-strain (20%), resulted in a 3-fold increase in both the ultimate tensile strength and modulus of ADCs. Similar mechanical activation of human mesenchymal stem cell (MSC) seeded ADCs in serum- and growth factor-free medium induced their tenogenic differentiation. Both static and cyclic loading profiles resulted in a greater than 12-fold increase in scleraxis gene expression and either suppressed or maintained osteogenic and chondrogenic expressions. Following the 48 h mechanoactivation period, the MSC-seeded scaffolds were matured by tethering in basal medium without further external mechanical stimulation for 19 days, altogether making up 21 days of culture. Extensive cell-induced matrix remodeling and deposition of collagen types I and III, tenascin-C and tenomodulin were observed, where initial cyclic loading induced significantly higher tenomodulin protein content. Moreover, the initial short-term mechanical stimulation elongated and polarized seeded MSCs, and overall cell alignment was significantly increased in those under static loading. These findings indicate the regenerative potential of the ADC scaffolds for short-term mechanoactivated tenogenic differentiation, which were achieved even in the absence of serum and growth factors that may potentially increase clinical translatability.

Effect of Menin Deletion in Early Osteoblast Lineage on the Mineralization of an In Vitro 3D Osteoid-like Dense Collagen Gel Matrix.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.

In Vitro Evaluation of Real-Time Viscoelastic and Coagulation Properties of Various Classes of Topical Hemostatic Agents Using a Novel Contactless Nondestructive Technology.

Hemorrhaging is the main cause of death among combat and civilian injuries and has significant clinical and economic consequences. Despite their vital roles in bleeding management, an optimal topical hemostatic agent (HA) has yet to be developed for a particular scenario. This is partly due to a lack of an overarching quantitative testing technology to characterize the various classes of HAs in vitro. Herein, the feasibility of a novel, contactless, and nondestructive technique to quantitatively measure the shear storage modulus (G') and clotting properties of whole blood in contact with different dosages of eight topical HAs, including particulates and gauze-like and sponge-like systems, was assessed. The real-time G'-time profiles of these blood/HA systems revealed their distinct biomechanical behavior to induce and impact coagulation. These were analyzed to characterize the clot initiation time, clotting rate, clotting time, and apparent stiffness of the formed clots (both immediately and temporally), which were correlated with their reported hemostatic mechanisms of action. Moreover, the HAs that worked independently from the natural blood clotting cascade were identified and quantified through this technology. In sum, this study indicated that the nondestructive nature of the technology may offer a promising tool for accurate, quantitative in vitro measurements of the clotting properties of various classes of HAs, which may be used to better predict their in vivo outcomes.

Osteoid-mimicking dense collagen/chitosan hybrid gels.

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.

Effect of phosphate-based glass fibre surface properties on thermally produced poly(lactic acid) matrix composites.

Incorporation of soluble bioactive glass fibres into biodegradable polymers is an interesting approach for bone repair and regeneration. However, the glass composition and its surface properties significantly affect the nature of the fibre-matrix interface and composite properties. Herein, the effect of Si and Fe on the surface properties of calcium containing phosphate based glasses (PGs) in the system (50P(2)O(5)-40CaO-(10-x)SiO(2)-xFe(2)O(3), where x = 0, 5 and 10 mol.%) were investigated. Contact angle measurements revealed a higher surface energy, and surface polarity as well as increased hydrophilicity for Si doped PG which may account for the presence of surface hydroxyl groups. Two PG formulations, 50P(2)O(5)-40CaO-10Fe(2)O(3) (Fe10) and 50P(2)O(5)-40CaO-5Fe(2)O(3)-5SiO(2) (Fe5Si5), were melt drawn into fibres and randomly incorporated into poly(lactic acid) (PLA) produced by melt processing. The ageing in deionised water (DW), mechanical property changes in phosphate buffered saline (PBS) and cytocompatibility properties of these composites were investigated. In contrast to Fe10 and as a consequence of the higher surface energy and polarity of Fe5Si5, its incorporation into PLA led to increased inorganic/organic interaction indicated by a reduction in the carbonyl group of the matrix. PLA chain scission was confirmed by a greater reduction in its molecular weight in PLA-Fe5Si5 composites. In DW, the dissolution rate of PLA-Fe5Si5 was significantly higher than that of PLA-Fe10. Dissolution of the glass fibres resulted in the formation of channels within the matrix. Initial flexural strength was significantly increased through PGF incorporation. After PBS ageing, the reduction in mechanical properties was greater for PLA-Fe5Si5 compared to PLA-Fe10. MC3T3-E1 preosteoblasts seeded onto PG discs, PLA and PLA-PGF composites were evaluated for up to 7 days indicating that the materials were generally cytocompatible. In addition, cell alignment along the PGF orientation was observed showing cell preference towards PGF.

Acellular dense collagen-S53P4 bioactive glass hybrid gel scaffolds form more bone than stem cell delivered constructs.

Dense collagen (DC) gels facilitate the osteoblastic differentiation of seeded dental pulp stem cells (DPSCs) and undergo rapid acellular mineralization when incorporated with bioactive glass particles, both in vitro and subcutaneously in vivo. However, the potential of DC-bioactive glass hybrid gels in delivering DPSCs for bone regeneration in an osseous site has not been investigated. In this study, the efficacies of both acellular and DPSC-seeded DC-S53P4 bioactive glass [(53)SiO2-(23)Na2O-(20)CaO-(4)P2O5, wt%] hybrid gels were investigated in a critical-sized murine calvarial defect. The incorporation of S53P4, an osteostimulative bioactive glass, into DC gels led to its accelerated acellular mineralization in simulated body fluid (SBF), in vitro, where hydroxycarbonated apatite was detected within 1 day. By day 7 in SBF, micro-mechanical analysis demonstrated an 8-fold increase in the compressive modulus of the mineralized gels. The in-situ effect of the bioactive glass on human-DPSCs within DC-S53P4 was evident, by their osteogenic differentiation in the absence of osteogenic supplements. The production of alkaline phosphatase and collagen type I was further increased when cultured in osteogenic media. This osteostimulative effect of DC-S53P4 constructs was confirmed in vivo, where after 8 weeks implantation, both acellular scaffolds and DPSC-seeded DC-S53P4 constructs formed mineralized and vascularized bone matrices with osteoblastic and osteoclastic cell activity. Surprisingly, however, in vivo micro-CT analysis confirmed that the acellular scaffolds generated larger volumes of bone, already visible at week 3 and exhibiting superior trabecular architecture. The results of this study suggest that DC-S53P4 scaffolds negate the need for stem cell delivery for effective bone tissue regeneration and may expedite their path towards clinical applications.

Three-dimensional mineralization of dense nanofibrillar collagen-bioglass hybrid scaffolds.

Scaffolds for bone tissue engineering must meet a number of requirements such as biocompatibility, osteoconductivity, osteoinductivity, biodegradability, and appropriate biomechanical properties. A combination of type I collagen and 45S5 Bioglass may meet these requirements, however, little has been demonstrated on the effect of Bioglass on the potential of the collagen nanofibrillar three-dimensional mineralization and its influence on the structural and mechanical properties of the scaffolds. In this work, rapidly fabricated dense collagen-Bioglass hybrid scaffolds were assessed for their potential for immediate implantation. Hybrid scaffolds were conditioned, in vitro, in simulated body fluid (SBF) for up to 14 days and assessed in terms of changes in structural, chemical, and mechanical properties. MicroCT and SEM analyses showed a homogeneous distribution of Bioglass particles in the as-made hybrids. Mineralization was detected at day 1 in SBF, while ATR-FTIR microscopy and XRD revealed the presence of hydroxyl-carbonated apatite on the surface and within the two hybrid scaffolds at days 7 and 14. FTIR and SEM confirmed that the triple helical structure and typical banding pattern of fibrillar collagen was maintained as a function of time in SBF. Principal component analysis executed on ATR-FTIR microscopy revealed that the mineralization extent was a function of both Bioglass content and conditioning time in SBF. Tensile mechanical analysis showed an increase in the elastic modulus and a corresponding decrease in strain at ultimate tensile strength (UTS) as imparted by mineralization of scaffolds as a function of time in SBF and Bioglass content. Change in UTS was affected by Bioglass content. These results suggested the achievement of a hybrid matrix potentially suitable for bone tissue engineering.

A nondestructive contactless technique to assess the viscoelasticity of blood clots in real-time.

There is a need for reliable and quantitative real-time assessment of blood properties to study and treat a broad spectrum of disorders and cardiovascular diseases as well as to test the efficacy of hemostatic agents. In this study, the real-time changes in viscoelastic/rheological properties of bovine whole blood during coagulation induced by different concentrations of calcium chloride (CaCl2; 15, 25, 35 and 45 mM) was investigated. For this purpose, a novel, contactless technique was used to accurately measure the clotting characteristics under controlled and sterile conditions. It was demonstrated that, increasing the calcium concentration from low values (i.e., 15 and 25 mM), led to shorter reaction time; however, a further increase in calcium concentration (i.e., 35 and 45 mM) favored longer reaction times. Additionally, increasing the CaCl2 concentration resulted in higher shear storage modulus (i.e., stiffer clots). These results were also comparable to those generated by thromboelastrograph, a clinically established technique, as well as a conventional rheometer, which quantitatively verified the high correlation of the shear storage modulus data. In sum, the non-destructive testing technique used in this study is reproducible and sensitive in measuring clot formation kinetics, which could be applied to assess the efficacy of hemostatic agents, and may also contribute to better diagnosing relevant circulatory system diseases and conditions.

Rapidly formed stable and aligned dense collagen gels seeded with Schwann cells support peripheral nerve regeneration.

OBJECTIVE: Gel aspiration-ejection (GAE) has recently been developed for the rapid production of dense, anisotropic collagen gel scaffolds with adjustable collagen fibrillar densities. In this study, a GAE system was applied to produce aligned Schwann cells within a type-1 collagen matrix to generate GAE-engineered neural tissues (GAE-EngNT) for potential nerve tissue engineering applications. APPROACH: The stability and mechanical properties of the constructs were investigated along with the viability, morphology and distribution of Schwann cells. Having established the methodology to construct stable robust Schwann cell-loaded engineered neural tissues using GAE (GAE-EngNTs), the potential of these constructs in supporting and guiding neuronal regeneration, was assessed both in vitro and in vivo. MAIN RESULTS: Dynamic mechanical analysis strain and frequency sweeps revealed that the GAE-EngNT produced via cannula gauge number 16 G (∼1.2 mm diameter) exhibited similar linear viscoelastic behaviors to rat sciatic nerves. The viability and alignment of seeded Schwann cells in GAE-EngNT were maintained over time post GAE, supporting and guiding neuronal growth in vitro with an optimal cell density of 2.0 × 106 cells ml-1. An in vivo test of the GAE-EngNTs implanted within silicone conduits to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that the constructs significantly promoted axonal regeneration and vascularization across the gap, as compared with the empty conduits although less effective regeneration compared with the autograft groups. SIGNIFICANCE: Therefore, this is a promising approach for generating anisotropic and robust engineered tissue which can be used with Schwann cells for peripheral nerve repair.

Viscoelastic and chemical properties of dentine after different exposure times to sodium hypochlorite, ethylenediaminetetraacetic acid and calcium hydroxide.

This study aims to evaluate the viscoelastic and chemical properties of dentine after different durations of exposure to 5.25% NaOCl, 17% EDTA and Ca(OH)2 solutions, and NaOCl in alternating combination with EDTA. Standard dentine bars were randomly assigned to: (i) formal-saline control-1; (ii) NaOCl; (iii) EDTA; (iv) NaOCl/EDTA; (v) formal-saline control-2; (vi) Ca(OH)2 pH 12.6; and (vii) Ca(OH)2 pH 9.8. Groups 1--4 underwent 10 min cycles of soaking and dynamic mechanical analysis up to 120 min. Groups 5-7 underwent similar tests at days 7, 14, 28 and 84. FTIR spectra of dentine discs exposed to the same regimens assessed surface chemistry. NaOCl or Ca(OH)2 (pH 12.6) solutions reduced the organic (N-H[1], N-H[3], C=0) peak components of dentine. This study demonstrated that accumulative damage of dentine could be facilitated by alternated exposure to NaOCl and EDTA. Exposure of dentine to Ca(OH)2 (pH12.6) for 7 days reduced viscous behaviour, inferring increased potential for fatigue failure.

Poly(d,l-Lactic acid) Composite Foams Containing Phosphate Glass Particles Produced via Solid-State Foaming Using CO2 for Bone Tissue Engineering Applications.

This study reports on the production and characterization of highly porous (up to 91%) composite foams for potential bone tissue engineering (BTE) applications. A calcium phosphate-based glass particulate (PGP) filler of the formulation 50P2O5-40CaO-10TiO2 mol.%, was incorporated into biodegradable poly(d,l-lactic acid) (PDLLA) at 5, 10, 20, and 30 vol.%. The composites were fabricated by melt compounding (extrusion) and compression molding, and converted into porous structures through solid-state foaming (SSF) using high-pressure gaseous carbon dioxide. The morphological and mechanical properties of neat PDLLA and composites in both nonporous and porous states were examined. Scanning electron microscopy micrographs showed that the PGPs were well dispersed throughout the matrices. The highly porous composite systems exhibited improved compressive strength and Young's modulus (up to >2-fold) and well-interconnected macropores (up to ~78% open pores at 30 vol.% PGP) compared to those of the neat PDLLA foam. The pore size of the composite foams decreased with increasing PGPs content from an average of 920 µm for neat PDLLA foam to 190 µm for PDLLA-30PGP. Furthermore, the experimental data was in line with the Gibson and Ashby model, and effective microstructural changes were confirmed to occur upon 30 vol.% PGP incorporation. Interestingly, the SSF technique allowed for a high incorporation of bioactive particles (up to 30 vol.%-equivalent to ~46 wt.%) while maintaining the morphological and mechanical criteria required for BTE scaffolds. Based on the results, the SSF technique can offer more advantages and flexibility for designing composite foams with tunable characteristics compared to other methods used for the fabrication of BTE scaffolds.

3D Printed Polyurethane Scaffolds for the Repair of Bone Defects.

Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection, and surgical resection of tumors. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modeling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralized matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularized fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralized tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.

Incorporation of vitamin E in poly(3hydroxybutyrate)/Bioglass composite films: effect on surface properties and cell attachment.

This study investigated the possibility of incorporating alpha-tocopherol (vitamin E) into poly(3hydroxybutyrate) (P(3HB))/Bioglass composites, which are being developed for bone tissue engineering matrices. P(3HB) films with 20 wt% Bioglass and 10 wt% vitamin E were prepared using the solvent casting technique. Addition of vitamin E significantly improved the hydrophilicity of the composites along with increasing the total protein adsorption. The presence of protein adsorbed on the composite surface was further confirmed using X-ray photoelectron spectroscopy analysis. Preliminary cell culture studies using MG-63 human osteoblasts showed that the addition of vitamin E in the P(3HB)/20 wt% Bioglass films significantly increased cell proliferation. The results achieved in this study confirmed the possibility of incorporating vitamin E as a suitable additive in P(3HB)/Bioglass composites to engineer the surface of the composites by promoting higher protein adsorption and increasing the hydrophilicity.

Bioinspired mineralization of a functionalized injectable dense collagen hydrogel through silk sericin incorporation.

Collagen based hydrogels are frequently used as templates to mimic the native biomineralization process. However, a lack of structural control and their inherently poor mineralization capability represent challenges when used as bone-extracellular-matrix mimicking constructs. The aspiration-ejection of highly-hydrated collagen gels allows for their densification and fibrillar remodelling, leading to the production of injectable dense collagen (I-DC) gel scaffolds characterized by an osteoid-like structure. In this study, silk-extracted sericin (SS), a negatively-charged protein that is rich in anionic amino-acids such as Asp and Glu, was hybridized into I-DC gels to induce hydroxyapatite deposition and stimulate the osteoblastic differentiation of seeded mesenchymal stem cells (MSCs). The effect of SS content on the acellular mineralization of I-DC gels in simulated body fluid (SBF) and on modulating the proliferation and osteogenesis of seeded MSCs, in vitro, were investigated. Methylene blue staining indicated increasingly negatively charged gels through SS incorporation. Attributable to the carboxyl groups provided by the acidic SS amino-acids, serving as calcium-phosphate nucleation sites, there was a time dependent increase in hydroxyapatite deposition, approaching 90 wt% by day 14 in SBF. Three dimensionally seeded MSCs attached and proliferated in all gel types and SS-incorporation led to an increase in their metabolic activity. Relative to neat I-DC gels, alkaline phosphatase (at day 7), runt related transcription factor 2 (at day 21) and osteocalcin (at days 14 and 21) expression was higher in MSCs when seeded in SS-incorporated I-DC gels. Cell-induced mineralization was accelerated in SS-incorporated I-DC gels suggesting its osteostimulative potential. In sum, SS incorporation into clinically relevant I-DC gels can provide a strategy to design scaffolds with potential applications in bone tissue engineering.

Effect of cell density on osteoblastic differentiation and matrix degradation of biomimetic dense collagen scaffolds.

Plastic compression of hyperhydrated collagen gels produces tissue-like scaffolds of enhanced biomechanical properties. By increasing collagen density, these scaffolds could be developed into highly Biomimetic cell-seeded templates. When utilizing three-dimensional (3-D) scaffold systems for tissue repair, and indeed when investigating the cytocompatibility of two-dimensional (2-D) surfaces, the cell seeding density is often overlooked. In this study, we investigated this potentially critical parameter using MG-63 cells seeded in the dense collagen scaffolds. This is conducted within the overall scope of developing these scaffolds for bone repair. Cell proliferation, osteoblastic differentiation, and matrix remodelling capacity in relation to various seeding densities, ranging from 10(5) to 10(8) cells/ml compressed collagen, were evaluated in vitro. This was performed using the AlamarBlue assay, quantitative polymerase chain reaction (qPCR), and tensile mechanical analysis respectively. Variations in cell seeding density significantly influenced cell proliferation where lower initial seeding density resulted in higher proliferation rates as a function of time in culture. Gene transcription levels for alkaline phosphatase (ALPL), runt-related transcription factor 2 (RUNX2), and osteonectin (SPARC) were also found to be dependent on the cell density. While ALPL transcription was down-regulated with culturing time for all seeding densities, there was an increase in RUNX2 and SPARC transcription, particularly for scaffolds with cell densities in the range 10(6)-10(7) cells/ml collagen. Furthermore, this range of seeding density affected cell capacity in conducting collagenous matrix degradation as established by analyzing matrix metalloproteinase 1 (MMP1) transcription and scaffold mechanical properties. This study has shown that the seeded cell population in the three-dimensional dense collagen scaffolds clearly affected the degree of osteoblastic cell proliferation, differentiation, and some aspects of matrix remodelling activity. The seeding density played a major role in influencing the corresponding cell differentiation and cell-matrix interaction.

Matrix mechanical properties of transversalis fascia in inguinal herniation as a model for tissue expansion.

Inguinal herniation represents a common condition requiring surgical intervention. Despite being regarded as a connective tissue disorder of uncertain cause, research has focused predominantly on biochemical changes in the key tissue layer, the transversalis fascia (TF) with little direct analysis of functional tissue mechanics. Connective tissue tensile properties are dominated by collagen fibril density and architecture. This study has correlated mechanical properties of herniated TF (HTF) and non-herniated TF (NHTF) with fibrillar properties at the ultrastructural level by quasi-static tensile mechanical analysis and image analysis of collagen electron micrographs. No significant difference was found between any of the key mechanical properties (break stress, strain or modulus) for HTF and NHTF. In addition, no significant differences were found in average collagen fibril diameter, density or fibre bundle spacing. However, both groups displayed anisotropy with greater break stress (p=0.001) on average in the transverse anatomical plane compared to the longitudinal plane in a mean ratio of 2:1 (anisotropy ratio), though there was no evidence of a difference in this ratio for HTF and NHTF for both break stress and modulus. It was noted that this anisotropy ratio corresponds closely with the expected force distribution on a model cylindrical structure loaded axially. The absence of other functional differences does not support the idea of a failing (injured) tissue but is consistent with it being a tissue undergoing chronic growth/expansion under multi-vectored mechanical loading. These findings provide new clues to collagen tissue herniation for mathematical modelling and model tissue engineering.