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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A Three-Dimensional Dense Collagen Hydrogel to Model Cancer Cell/Osteoblast Interactions.

No curative treatment options exist once breast cancer metastasizes to bone. This is due, in part, to an incomplete understanding of how osteolytic cancers interact with bone. Presented here is a novel approach to study the interactions between triple negative breast cancer cells and osteoblasts within a 3D collagenous environment. More specifically, a dense collagen hydrogel was employed to model interactions between MDA-MB-231 breast cancer cells and MC3T3-E1 pre-osteoblasts. Co-cultures with these two cell types, or MDA-MB-231-derived conditioned medium applied to MC3T3-E1 cells, were established in the context of plastically compressed dense collagen gel matrices. Importantly, breast cancer-derived conditioned medium or the establishment of breast cancer/osteoblast co-cultures did not negatively influence MC3T3-E1 cell viability. The inclusion of either conditioned medium or the presence of MDA-MB-231 cells resulted in impaired MC3T3-E1 differentiation into osteoblasts, which coincided with reduced osteoblast-mediated mineralization. The results presented here demonstrate that dense collagen gels provide a model environment to examine the effect of osteolytic breast cancer cells on osteoblast differentiation and subsequent mineralization of the collagen scaffold.

Functionalization of silk fibroin through anionic fibroin derived polypeptides.

While silk fibroin (SF)-based fibrous matrices are often considered as templates to mimic the native biomineralization process, their limited ability to induce apatite deposition hinders their potential applications in bone tissue engineering. In this study, it was hypothesized that the incorporation of anionic fibroin derived polypeptides (Cs), generated through the α-chymotrypsin digestion of SF, into SF would induce apatite deposition. The effect of Cs incorporation and content on the mineralization of fibrous, electrospun (ES) SF matrices, was assessed in simulated body fluid (SBF). Moreover, the potential role of Cs in mediating the proliferation and osteoblastic differentiation of seeded mesenchymal stem cells (MSCs), in vitro, was also investigated. Methylene blue staining indicated that the ES SF matrices became increasingly negatively charged with an increase in Cs content. Furthermore, the mechanical properties of the ES SF matrices were modulated through variations in Cs content. Their subsequent immersion in SBF demonstrated rapid mineralization, attributable to the carboxyl groups provided by the negatively charged Cs polypeptides, which served as nucleation sites for apatite deposition. Seeded MSCs attached on all scaffold types with differences observed in metabolic activities when cultured in osteogenic medium. Relative to basal medium, there was an up-regulation of alkaline phosphatase, runt related transcription factor 2 and osteocalcin in osteogenic medium (at days 14 and 21). Cell-induced mineralized matrix deposition appeared to be accelerated on Cs incorporated ES SF suggesting an osteoinductive potential of these polypeptides. In sum, the ability to incorporate Cs into SF scaffolds offers promise in bone tissue engineering applications.

Zein-based composites in biomedical applications.

Considerable research efforts have been devoted to zein-based biomaterials for tissue engineering and other biomedical applications over the past decade. The attention given to zein-based polymers is primarily attributed to their biocompatibility and biodegradability. However, due to the relatively low mechanical properties of these polymers, numerous inorganic compounds (e.g., hydroxyapatite, calcium phosphate, bioactive glasses, natural clays) have been considered in combination with zein to create composite materials in an attempt to enhance zein mechanical properties. Inorganic phases also positively impact on the hydrophilic properties of zein matrices inducing a suitable environment for cell attachment, spreading, and proliferation. This review covers available literature on zein and zein-based composite materials, with focus on the combination of zein with commonly used inorganic fillers for tissue engineering and drug delivery applications. An overview of the most recent advances in fabrication techniques for zein-based composites is presented and key applications areas and future developments in the field are highlighted. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1656-1665, 2017.

Multilayered dense collagen-silk fibroin hybrid: a platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineages.

Type I collagen is a major structural and functional protein in connective tissues. However, collagen gels exhibit unstable geometrical properties, arising from extensive cell-mediated contraction. In an effort to stabilize collagen-based hydrogels, plastic compression was used to hybridize dense collagen (DC) with electrospun silk fibroin (SF) mats, generating multilayered DC-SF-DC constructs. Seeded mesenchymal stem cell (MSC)-mediated DC-SF-DC contraction, as well as growth and differentiation under chondrogenic and osteogenic supplements, were compared to those seeded in DC and on SF alone. The incorporation of SF within DC prevented extensive cell-mediated collagen gel contraction. The effect of the multilayered hybrid on MSC remodelling capacity was also evident at the transcription level, where the expression of matrix metalloproteinases and their inhibitor (MMP1, MMP2, MMP3, MMP13 and Timp1) by MSCs within DC-SF-DC were comparable to those on SF and significantly downregulated in comparison to DC, except for Timp1. Chondrogenic supplements stimulated extracellular matrix production within the construct, stabilizing its multilayered structure and promoting MSC chondrogenic differentiation, as indicated by the upregulation of the genes Col2a1 and Agg and the production of collagen type II. In osteogenic medium there was an upregulation in ALP and OP along with the presence of an apatitic phase, indicating MSC osteoblastic differentiation and matrix mineralization. In sum, these results have implications on the modulation of three-dimensional collagen-based gel structural stability and on the stimulation and maintenance of the MSC committed phenotype inherent to the in vitro formation of chondral tissue and bone, as well as on potential multilayered complex tissues. Copyright © 2015 John Wiley & Sons, Ltd.

Bioactive glasses in wound healing: hope or hype?

Bioactive glasses have long been investigated in mineralized tissue regeneration, but recently their potential applications in soft tissue repair, and in particular wound healing, have demonstrated great promise. Commonly used glasses, such as the silicate-based Bioglass® 45S5 [(46.1)SiO2-(26.9)CaO-(24.4)Na2O-(2.6)P2O5 (mol%)] and borate-based 13-93B3 [(54)B2O3-(22)CaO-(6)Na2O-(8)K2O-(8)MgO-(2)P2O5 (mol%)] have been implicated in the stages of wound healing due to their ability to release ions that can stimulate processes, such as haemostasis, antibacterial efficacy, epithelial cell migration, angiogenesis, and fibroblastic cell proliferation, amongst others. More recently, a wound dressing composed of a borate-based glass received regulatory approval for use in the treatment of acute and chronic wounds. However, to date, there are no comprehensive reports on their specific mechanism of action in accelerating the wound healing processes. In this highlight, we will provide a brief overview of the wound healing stages, review the bioactive glass formulations that have been investigated for potential applications in wound healing and attempt to summarize the consensus on why these glasses may be successful in wound healing.

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells.

Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.

A gel aspiration-ejection system for the controlled production and delivery of injectable dense collagen scaffolds.

A gel aspiration-ejection (GAE) system has been developed for the advanced production and delivery of injectable dense collagen (I-DC) gels of unique collagen fibrillar densities (CFDs). Through the creation of negative pressure, GAE aspirates prefabricated highly hydrated collagen gels into a needle, simultaneously inducing compaction and meso-scale anisotropy (i.e., fibrillar alignment) on the gels, and by subsequent reversal of the pressure, I-DC gels can be controllably ejected. The system generates I-DC gels with CFDs ranging from 5 to 32 wt%, controlling the initial scaffold microstructure, anisotropy, hydraulic permeability, and mechanical properties. These features could potentially enable the minimally invasive delivery of more stable hydrogels. The viability, metabolic activity, and differentiation of seeded mesenchymal stem cells (MSCs) was investigated in the I-DC gels of distinct CFDs and extents of anisotropy produced through two different gauge needles. MSC osteoblastic differentiation was found to be relatively accelerated in I-DC gels that combined physiologically relevant CFDs and increased fibrillar alignment. The ability to not only support homogenous cell seeding, but also to direct and accelerate their differentiation through tissue-equivalent anisotropy, creates numerous opportunities in regenerative medicine.