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.

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.

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.

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.

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.

Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery.

Fabrication of injectable, cellular, anisotropic collagen tissue equivalents with modular fibrillar densities.

Technological improvements in collagen gel fabrication are highly desirable as they may enable significant advances in the formation of tissue-equivalent biomaterials for regenerative medicine, three-dimensional (3D) in vitro tissue models, and injectable scaffolds for cell and drug delivery applications. Thus, strategies to modulate collagen gel fibrillar density and organization in the mesostructure have been pursued to fabricate collagenous matrices with extracellular matrix-like features. Herein, we introduce a robust and simple method, namely gel aspiration-ejection (GAE), to engineer 3D, anisotropic, cell seeded, injectable dense collagen (I-DC) gels with controllable fibrillar densities, without the use of crosslinking. GAE allows for the hybridization of collagen gels with bioactive agents for increased functionality and supports highly aligned homogenous cell seeding, thus providing I-DC gels with distinct properties when compared to isotropic DC gels of random fibrillar orientation. The hybridization of I-DC with anionic fibroin derived polypeptides resulted in the nucleation of carbonated hydroxyapatite within the aligned nanofibrillar network upon exposure to simulated body fluid, yielding a 3D, anisotropic, mineralized collagen matrix. In addition, I-DC gels accelerated the osteoblastic differentiation of seeded murine mesenchymal stem cells (m-MSCs) when exposed to osteogenic supplements, which resulted in the cell-mediated, bulk mineralization of the osteoid-like gels. In addition, and upon exposure to neuronal transdifferentiation medium, I-DC gels supported and accelerated the differentiation of m-MSCs toward neuronal cells. In conclusion, collagen GAE presents interesting opportunities in a number of fields spanning tissue engineering and regenerative medicine to drug and cell delivery.

The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen-silk fibroin construct.

Airway tracts serve as a conduit of transport in the respiratory system. Architecturally, these are composed of cartilage rings that offer flexibility and prevent collapse during normal breathing. To this end, the successful regeneration of an airway tract requires the presence of differentiated chondrocytes and airway smooth muscle cells. This study investigated the role of physiological dynamic mechanical stimulation, in vitro, on the differentiation of mesenchymal stem cells (MSCs), three-dimensionally seeded within a tubular dense collagen matrix construct-reinforced with rings of electrospun silk fibroin mat (TDC-SFC). In particular, the role of either shear stress supplied by laminar fluid flow or cyclic shear stress in combination with circumferential strain, provided by pulsatile flow, on the chondrogenic differentiation, and contractile lineage of MSCs, and their effects on TDC-SFC morphology and mechanical properties were analysed. Chondrogenic differentiation of MSCs was observed in the presence of chondrogenic supplements under both static and laminar flow cultures. In contrast, physiological pulsatile flow resulted in preferential cellular orientation within TDC-SFC, as dictated by dynamic circumferential strain, and induced MSC contractile phenotype expression. In addition, pulsatile flow decreased MSC-mediated collagen matrix remodelling and increased construct circumferential strength. Therefore, TDC-SFC demonstrated the central role of a matrix in the delivery of mechanical stimuli over chemical factors, by providing an in vitro niche to control MSC differentiation, alignment and its capacity to remodel the matrix.

Characterization of aqueous interactions of copper-doped phosphate-based glasses by vapour sorption.

Owing to their adjustable dissolution properties, phosphate-based glasses (PGs) are promising materials for the controlled release of bioinorganics, such as copper ions. This study describes a vapour sorption method that allowed for the investigation of the kinetics and mechanisms of aqueous interactions of PGs of the formulation 50P2O5-30CaO-(20-x)Na2O-xCuO (x=0, 1, 5 and 10mol.%). Initial characterization was performed using (31)P magic angle spinning nuclear magnetic resonance and attenuated total reflectance-Fourier transform infrared spectroscopy. Increasing CuO content resulted in chemical shifts of the predominant Q(2) NMR peak and of the (POP)as and (PO(-)) Fourier transform infrared absorptions, owing to the higher strength of the POCu bond compared to PONa. Vapour sorption and desorption were gravimetrically measured in PG powders exposed to variable relative humidity (RH). Sorption was negligible below 70% RH and increased exponentially with RH from 70 to 90%, where it exhibited a negative correlation with CuO content. Vapour sorption in 0% and 1% CuO glasses resulted in phosphate chain hydration and hydrolysis, as evidenced by protonated Q(0)(1H) and Q(1)(1H) species. Dissolution rates in deionized water showed a linear correlation (R(2)>0.99) with vapour sorption. Furthermore, cation release rates could be predicted based on dissolution rates and PG composition. The release of orthophosphate and short polyphosphate species corroborates the action of hydrolysis and was correlated with pH changes. In conclusion, the agreement between vapour sorption and routine characterization techniques in water demonstrates the potential of this method for the study of PG aqueous reactions.

Silk fibroin derived polypeptide-induced biomineralization of collagen.

Silk fibroin (SF) is extensively investigated in osteoregenerative therapy as it combines extraordinary mechanical properties and directs calcium-phosphate formation. However, the role of the peptidic fractions in inducing the protein mineralization has not been previously decoded. In this study, we investigated the mineralization of fibroin-derived polypeptides (FDPs), which were obtained through the chymotryptic separation of the hydrophobic crystalline (Cp) fractions and of the hydrophilic electronegative amorphous (Cs) fractions. When immersed in simulated body fluid (SBF), only Cs fragments demonstrated the formation of carbonated apatite, providing experimental evidence that the mineralization of SF is dictated exclusively by its electronegative amino-acidic sequences. The potential of Cs to conceptually mimic the role of anionic non-collagenous proteins in biomineralization processes was investigated via their incorporation (up to 10% by weight) in bulk osteoid-like dense collagen (DC) gels. Within 6 h in SBF, apatite was formed in DC-Cs hybrid gels, and by day 7, carbonated hydroxylapatite crystals were extensively formed. This accelerated 3-D mineralization resulted in a nine-fold increase in the compressive modulus of the hydrogel. The tailoring of the mineralization and mechanical properties of hydrogels through hybridization with FDPs could potentially have a significant impact on cell delivery and bone regenerative medicine.

Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications.

Tissue engineering of multilayered constructs that model complex tissues poses a significant challenge for regenerative medicine. In this study, a three-layered scaffold consisting of an electrospun silk fibroin (SF) mat sandwiched between two dense collagen (DC) layers was designed and characterized. It was hypothesized that the SF layer would endow the DC-SF-DC construct with enhanced mechanical properties (e.g., apparent modulus, tensile strength, and toughness), while the surrounding DC layers provide an extracellular matrix-like environment for mesenchymal stem cell (MSC) growth. MSC-seeded DC-SF-DC hybrids were produced using the plastic compression technique and characterized morphologically, chemically, and mechanically. Moreover, MSC viability was assessed for up to 1 wk in culture. Scaffold analyses confirmed compaction and integration of the meso-scaled multilayered DC-SF-DC hybrid, which was reflected in a significantly higher toughness value when compared to DC and SF alone. MSCs directly incorporated into the DC layers remained viable for up to day 7. The ease of multilayered construct fabrication, enhanced biomechanical properties, along with uniformity of cell distribution confirmed the possibility for the incorporation and segregation of different cell types within distinct layers for the regeneration of complex tissues, such as skin, or central nervous system dura mater.

Reduced hydraulic permeability of three-dimensional collagen scaffolds attenuates gel contraction and promotes the growth and differentiation of mesenchymal stem cells.

Optimal scaffold characteristics are essential for the therapeutic application of engineered tissues. Hydraulic permeability (k) affects many properties of collagen gels, such as mechanical properties, cell-scaffold interactions within three dimensions (3D), oxygen flow and nutrient diffusion. However, the cellular response to 3D gel scaffolds of defined k values has not been investigated. In this study, unconfined plastic compression under increasing load was used to produce collagen gels with increasing solid volume fractions. The Happel model was used to calculate the resulting permeability values in order to study the interaction of k with gel mechanical properties and mesenchymal stem cell (MSC)-induced gel contraction, metabolism and differentiation in both non-osteogenic (basal medium) and osteogenic medium for up to 3 weeks. Collagen gels of fibrillar densities ranging from 0.3 to >4.1 wt.% gave corresponding k values that ranged from 1.00 to 0.03 microm(2). Mechanical testing under compression showed that the collagen scaffold modulus increased with collagen fibrillar density and a decrease in k value. MSC-induced gel contraction decreased as a direct function of decreasing k value. Relative to osteogenic conditions, non-osteogenic MSC cultures exhibited a more than 2-fold increase in gel contraction. MSC metabolic activity increased similarly under both osteogenic and non-osteogenic culture conditions for all levels of plastic compression. Under osteogenic conditions MSC differentiation and mineralization, as indicated by alkaline phosphatase activity and von Kossa staining, respectively, increased in response to an elevation in collagen fibrillar density and decreased gel permeability. In this study, gel scaffolds with higher collagen fibrillar densities and corresponding lower k values provided a greater potential for MSC differentiation and appear most promising for bone grafting purposes. Thus, cell-scaffold interactions can be optimized by defining the 3D properties of collagen scaffolds through k adjustment.

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.

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.

Lactic acid based PEU/HA and PEU/BCP composites: Dynamic mechanical characterization of hydrolysis.

Lactic acid based poly(ester-urethane) (PEU-BDI) and its composites with 20 and 40 vol.% bioceramic filler were characterized prior to their use as biocompatible and bioabsorbable artificial bone materials. Morphological, dynamic mechanical properties, and degradation of these either hydroxyapatite or biphasic calcium phosphate containing composites were determined. Addition of particulate bioactive filler increased the composite stiffness and the glass transition temperature, indicating strong interactions between the filler and matrix. Materials were sterilized by gamma-irradiation, which reduced the average molecular weights by 30-40%. However, dynamic mechanical properties were not significantly affected by irradiation. Specimens were immersed in 0.85 w/v saline at 37 degrees C for 5 weeks, and changes in molecular weights, mass, water absorption, and dynamic mechanical properties were recorded. All the composite materials showed promising dynamic mechanical performance over the 5 weeks of hydrolysis. Average molecular weights of PEU-BDI and its composites did not change substantially during the test period. PEU-BDI retained its modulus values relatively well, and although the moduli of the composite materials were much higher, especially at high filler content, they exhibited faster loss of mechanical integrity.