Nanostructured gellan and xanthan hydrogel depot integrated within a baghdadite scaffold augments bone regeneration

Controlled delivery of biological cues through synthetic scaffolds to enhance the healing capacity of bone defects is yet to be realized clinically. The purpose of this study was development of a bioactive tissue‐engineered scaffold providing the sustained delivery of an osteoinductive drug, dexamethasone disodium phosphate (DXP), encapsulated within chitosan nanoparticles (CN). Porous baghdadite (BD; Ca₃ZrSi₂O₉) scaffolds, a zirconia‐modified calcium silicate ceramic, was coated with DXP‐encapsulated CN nanoparticles (DXP–CN) using nanostructured gellan and xanthan hydrogel (GX). Crosslinker and GX polymer concentrations were optimized to achieve a homogeneous distribution of hydrogel coating within BD scaffolds. Dynamic laser scattering indicated an average size of 521 ± 21 nm for the DXP–CN nanoparticles. In vitro drug‐release studies demonstrated that the developed DXP–CN–GX hydrogel‐coated BD scaffolds (DXP–CN–GX–BD) resulted in a sustained delivery of DXP over the 5 days (78 ± 6% of drug release) compared with burst release over 1 h, seen from free DXP loaded in uncoated BD scaffolds (92 ± 8% release in 1 h). To estimate the influence of controlled delivery of DXP from the developed scaffolds, the effect on MG 63 cells was evaluated using various bone differentiation assays. Cell culture within DXP–CN–GX–BD scaffolds demonstrated a significant increase in the expression of early and late osteogenic markers of alkaline phosphatase activity, collagen type 1 and osteocalcin, compared to the uncoated BD scaffold. The results suggest that the DXP‐releasing nanostructured hydrogel integrated within the BD scaffold caused sustained release of DXP, improving the potential for osteogenic differentiation. Copyright © 2015 John Wiley & Sons, Ltd.     

Design,fabrication and in vitro evaluation of a novel polymer‐hydrogel hybrid scaffold for bone tissue engineering

The development of a bone mechanically‐compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically‐sized bone defects. Although previous studies with weight‐bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold‐based bone regeneration. In this study, we designed and fabricated a novel polymer‐hydrogel hybrid scaffold system in which a load‐bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre‐osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin‐streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre‐osteoblast cell line MC3T3‐E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx‐2 and osteocalcin (OC) increased in MC3T3‐E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer‐hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Polydopamine-modified poly(l-lactic acid) nanofiber scaffolds immobilized with an osteogenic growth peptide for bone tissue regeneration

It is highly desirable for bone tissue engineering scaffolds to have significant osteogenic properties and capability to improve cell growth and thus enhance bone regeneration. In this study, a poly(l-lactic acid) (PLLA) nanofiber scaffold-immobilized osteogenic growth peptide (OGP) was prepared via polydopamine (PDA) coating. X-ray photoelectron spectroscopy (XPS), contact angle measurement, and scanning electron microscopy (SEM) were used to determine the OGP immobilization, hydrophilicity and surface roughness of the samples. The SEM and fluorescence images demonstrate that the PLLA nanofiber scaffolds immobilized with the OGP have excellent cytocompatibility in terms of cell adhesion and proliferation. The ALP activity and the Runx2 and OPN expression results indicated that the PLLA nanofiber scaffolds immobilized with OGP significantly enhanced the osteogenic differentiation and calcium mineralization of hMSCs in vitro. A rat model of critical skull bone defect was selected to evaluate the bone formation capacity of the scaffolds. Micro CT analysis and histological results demonstrated that the PLLA scaffolds immobilized with OGP significantly promoted bone regeneration in critical-sized bone defects. This study verifies that the PLLA scaffold-immobilized OGP has significant potential in bone tissue engineering.

Polydopamine-modified PLLA nanofiber scaffolds immobilized with osteogenic growth peptide were designed and prepared for promoting bone formation.     

Fabrication of a biomimetic ZeinPDA nanofibrous scaffold impregnated with BMP‐2 peptide conjugated TiO2 nanoparticle for bone tissue engineering

A biomimetic Zein polydopamine based nanofiber scaffold was fabricated to deliver bone morphogenic protein‐2 (BMP‐2) peptide conjugated titanium dioxide nanoparticles in a sustained manner for investigating its osteogenic differentiation potential. To prolong its retention time at the target site, BMP‐2 peptide has been conjugated to titanium dioxide nanoparticles owing to its high surface to volume ratio. The effect of biochemical cues from BMP‐2 peptide and nanotopographical stimulation of electrospun Zein polydopamine nanofiber were examined for its enhanced osteogenic expression of human fetal osteoblast cells. The sustained delivery of bioactive signals, improved cell adhesion, mineralization, and differentiation could be attributed to its highly interconnected nanofibrous matrix with unique material composition. Further, the expression of osteogenic markers revealed that the fabricated nanofibrous scaffold possess better cell—biomaterial interactions. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for bone regeneration.     

Adipose‐ and bone marrow‐derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass‐based scaffolds

Mesenchymal stem cells can be isolated from a variety of different sources, each having their own peculiar merits and drawbacks. Although a number of studies have been conducted comparing these stem cells for their osteo‐differentiation ability, these are mostly done in culture plastics. We have selected stem cells from either adipose tissue (ADSCs) or bone marrow (BMSCs) and studied their differentiation ability in highly porous three‐dimensional (3D) 45S5 Bioglass®‐based scaffolds. Equal numbers of cells were seeded onto 5 × 5 × 4 mm³ scaffolds and cultured in vitro, with or without osteo‐induction medium. After 2 and 4 weeks, the cell–scaffold constructs were analysed for cell number, cell spreading, viability, alkaline phosphatase activity and osteogenic gene expression. The scaffolds with ADSCs displayed osteo‐differentiation even without osteo‐induction medium; however, with osteo‐induction medium osteogenic differentiation was further increased. In contrast, the scaffolds with BMSCs showed no osteo‐differentiation without osteo‐induction medium; after application of osteo‐induction medium, osteo‐differentiation was confirmed, although lower than in scaffolds with ADSCs. In general, stem cells in 3D bioactive glass scaffolds differentiated better than cells in culture plastics with respect to their ALP content and osteogenic gene expression. In summary, 45S5 Bioglass‐based scaffolds seeded with ADSCs are well‐suited for possible bone tissue‐engineering applications. Induction of osteogenic differentiation appears unnecessary prior to implantation in this specific setting. Copyright © 2013 John Wiley & Sons, Ltd.     

Loading of BMP‐2‐related peptide onto three‐dimensional nano‐hydroxyapatite scaffolds accelerates mineralization in critical‐sized cranial bone defects

Extrusion free‐forming, as a rapid prototyping technique, is extensively applied in fabricating ceramic material in bone tissue engineering. To improve the osteoinductivity of nano‐hydroxyapatite (nHA) scaffold fabricated by extrusion free‐forming, in this study, we incorporated a new peptide (P28) and optimized the superficial microstructure after shaping by controlling the sintering temperature. P28, a novel bone morphogenic protein 2 (BMP‐2)‐related peptide, was designed in this study. Analysis of the structure, physicochemical properties and release kinetics of P28 from nHA sintered at temperatures ranging from 1000 °C to 1400 °C revealed that nHA sintered at 1000 °C had higher porosity, preferable pore size and better capacity to control P28 release than that sintered at other temperatures. Moreover, the nHA scaffold sintered at 1000 °C with P28 showed improved adhesion, proliferation and osteogenic differentiation of MC3T3‐E1 cells compared with scaffolds lacking P28 or BMP‐2. In vivo, nHA scaffolds sintered at 1000 °C with P28 or BMP‐2 induced greater bone regeneration in critical‐sized rat cranial defects at 6 and 12 weeks post‐implantation compared with scaffolds lacking P28 or BMP‐2. Thus, nHA scaffolds sintered at 1000 °C and loaded with P28 may be excellent biomaterials for bone tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Design and biological functionality of a novel hybrid Ti‐6Al‐4V/hydrogel system for reconstruction of bone defects

We have designed a unique injectable bioactive hydrogel comprising of alginate, gelatin, and nanocrystalline hydroxyapatite and loaded with osteoblasts, with the ability to infiltrate into three‐dimensional Ti‐6Al‐4V scaffolds with interconnected porous architecture, fabricated by electron beam melting. A two‐step crosslinking process using the EDC/NHS and CaCl₂ was adopted and found to be effective in the fabrication of cell‐loaded hydrogel/Ti‐6Al‐4V scaffold system. This hybrid Ti‐6Al‐4V scaffold/hydrogel system was designed for the reconstruction of bone defects, which are difficult to heal in the absence of suitable support materials. The hybrid Ti‐6Al‐4V/hydrogel system favourably modulated the biological functions, namely, adhesion, proliferation, cell‐to‐cell, and cell‐material communication because of the presence of extracellular matrix‐like hydrogel in the interconnected porous structure of 3D printed Ti‐6Al‐4V scaffold. The hydrogel was cytocompatible, which was proven through live/dead assay, the expression level of prominent proteins for cell adhesion and cytoskeleton, including 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay. Furthermore, the high bone formation ability of the hydrogel was confirmed using alkaline phosphatase assay. A high equilibrium water content (~97%) in the hydrogel enables the delivery of cells and bioactive molecules, necessary for bone tissue growth. Although not studied, the presence of hydrogel in the pores of the scaffold can provide the space for the cell migration as well as vascularization through it, required for the effective exchange of nutrients. In conclusion, we underscore that the 3D‐printed Ti‐6Al‐4V scaffold‐loaded with bioactive hydrogel to treat the bone defects significantly impacted cellular functions and cell‐material interaction.     

Hybrid scaffolds of Mg alloy mesh reinforced polymer/extracellular matrix composite for critical‐sized calvarial defect reconstruction

The challenge of developing scaffolds to reconstruct critical‐sized calvarial defects without the addition of high levels of exogenous growth factor remains relevant. Both osteogenic regenerative efficacy and suitable mechanical properties for the temporary scaffold system are of importance. In this study, a Mg alloy mesh reinforced polymer/demineralized bone matrix (DBM) hybrid scaffold was designed where the hybrid scaffold was fabricated by a concurrent electrospinning/electrospraying of poly(lactic‐co‐glycolic acid) (PLGA) polymer and DBM suspended in hyaluronic acid (HA). The Mg alloy mesh significantly increased the flexural strength and modulus of PLGA/DBM hybrid scaffold. In vitro results demonstrated that the Mg alloy mesh reinforced PLGA/DBM hybrid scaffold (Mg‐PLGA@HA&DBM) exhibited a stronger ability to promote the proliferation of bone marrow stem cells (BMSCs) and induce BMSC osteogenic differentiation compared with control scaffolding materials lacking critical components. In vivo osteogenesis studies were performed in a rat critical‐sized calvarial defect model and incorporated a variety of histological stains and immunohistochemical staining of osteocalcin. At 12 weeks, the rat model data showed that the degree of bone repair for the Mg‐PLGA@HA&DBM scaffold was significantly greater than for those scaffolds lacking one or more of the principal components. Although complete defect filling was not achieved, the improved mechanical properties, promotion of BMSC proliferation and induction of BMSC osteogenic differentiation, and improved promotion of bone repair in the rat critical‐sized calvarial defect model make Mg alloy mesh reinforced PLGA/DBM hybrid scaffold an attractive option for the repair of critical‐sized bone defects where the addition of exogenous isolated growth factors is not employed.     

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright © 2008 John Wiley & Sons, Ltd.     

Injectable alendronate-functionalized GelMA hydrogels for mineralization and osteogenesis

In a minimally invasive procedure, hydrogels can be injected into an affected area for drug loading and tissue repair. Here, gelatin methacryloyl (GelMA) was modified with alendronate (ALN), and three different alendronate-functionalized GelMA (GelMA-ALN) hydrogels were prepared. The modification greatly improved the swelling ratio, protein adsorption and mineralization. The GelMA-ALN hydrogels significantly promoted the in vitro osteogenic differentiation of hFOB cells as indicated by their higher alkaline phosphatase (ALP) activity, denser mineralization and up-regulated osteogenesis-related genes at both the mRNA and protein levels. Meanwhile, the cells maintained their activity and differentiated into osteoblasts when encapsulated in the GelMA-ALN hydrogels. Alendronate-modified hydrogels have potential for use in the minimally invasive treatment of irregular bone defects.

Injectable alendronate-modified GelMA hydrogel greatly improved mineralization and in vitro osteogenesis both at the surface and inside of the hydrogel, which have potential in treatment of irregular bone defects.     

Behaviour of human mesenchymal stem cells on chemically synthesized HA–PCL scaffolds for hard tissue regeneration

Our goal was to characterize the response of human mesenchymal stem cells (hMSCs) to a novel composite scaffold for bone tissue engineering. The hydroxyapatite–polycaprolactone (HA–PCL) composite scaffolds were prepared by a sol–gel method at room temperature and the scaffold morphology was investigated by scanning electron microscopy (SEM)/energy‐dispersive spectroscopy (EDS) to validate the synthesis process. The response of two different lines of hMSCs, bone‐marrow‐derived human mesenchymal stem cells (BMSCs) and dental pulp stem cells (DPSCs) in terms of cell proliferation and differentiation into the osteoblastic phenotype, was evaluated using Alamar blue assay, SEM, histology and alkaline phosphatase activity. Our results indicate that tissue engineering by means of composite HA–PCL scaffolds may represent a new therapeutic strategy to repair craniofacial bone defects. Copyright © 2013 John Wiley & Sons, Ltd.     

Photopolymerizable chitosan–collagen hydrogels for bone tissue engineering

Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue‐engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi‐interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC–Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC–Col composite hydrogels may be useful in promoting bone regeneration. Copyright © 2014 John Wiley & Sons, Ltd.     

Tissue‐engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in femoral defects

This study aims to assess the in vivo performance of cell–scaffold constructs composed of goat marrow stromal cells (GBMCs) and SPCL (a blend of starch with polycaprolactone) fibre mesh scaffolds at different stages of development, using an autologous model. GBMCs from iliac crests were seeded onto SPCL scaffolds and in vitro cultured for 1 and 7 days in osteogenic medium. After 1 and 7 days, the constructs were characterized for proliferation and initial osteoblastic expression by alkaline phosphatase (ALP) activity. Scanning electron microscopy analysis was performed to investigate cellular morphology and adhesion to SPCL scaffolds. Non‐critical defects (diameter 6 mm, depth 3 mm) were drilled in the posterior femurs of four adult goats from which bone marrow and serum had been collected previously. Drill defects alone and defects filled with scaffolds without cells were used as controls. After implantation, intravital fluorescence markers, xylenol orange, calcein green and tetracycline, were injected subcutaneously after 2, 4 and 6 weeks, respectively, for bone formation and mineralization monitoring. Subsequently, samples were stained with Lévai–Laczkó for bone formation and histomorphometric analysis. GBMCs adhered and proliferated on SPCL scaffolds and an initial differentiation into pre‐osteoblasts was detected by an increasing level of ALP activity with the culture time. In vivo experiments indicated that bone neoformation occurred in all femoral defects. The results obtained provided important information about the performance of SPCL–GBMC constructs in an orthotopic goat model that enabled future studies to be designed to investigate in vivo the functionality of SPCL–GBMC constructs in more complex models, viz. critical sized defects, and to evaluate the influence of in vitro cultured autologous cells in the healing and bone regenerative process. Copyright © 2010 John Wiley & Sons, Ltd.     

Evaluating the feasibility of utilizing the small molecule phenamil as a novel biofactor for bone regenerative engineering

Osteoblast cell adhesion and differentiation on biomaterials are important achievements necessary for implants to be useful in bone regenerative engineering. Recombinant bone morphogenetic proteins (BMPs) have been shown to be important for these processes; however, there are many challenges associated with the widespread use of these proteins. A recent report demonstrated that the small molecule phenamil, a diuretic derivative, was able to induce osteoblast differentiation and mineralization in vitro via the canonical BMP signalling cascade (Park et al., 2009). In this study, the feasibility of using phenamil as a novel biofactor in conjunction with a biodegradable poly(lactide‐co‐glycolide acid) (PLAGA) polymeric scaffold for engineering bone tissue was evaluated. The in vitro cellular behaviour of osteoblast‐like MC3T3‐E1 cells cultured on PLAGA scaffolds in the presence of phenamil at 10 μM were characterized with regard to initial cell adhesion, proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. The results demonstrate that phenamil supported cell proliferation, promoted ALP activity and facilitated matrix mineralization of osteoblast‐like MC3T3‐E1 cells. Moreover, in this study, we found that phenamil promoted integrin‐mediated cell adhesion on PLAGA scaffolds. It was also shown that phenamil encapsulated within porous, microsphere PLAGA scaffolds retained its osteogenic activity upon release. Based on these findings, the small molecule phenamil has the potential to serve as a novel biofactor for the repair and regeneration of bone tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

Calcium deposition in photocrosslinked poly(Pro‐Hyp‐Gly) hydrogels encapsulated rat bone marrow stromal cells

Reproducing the features of the extracellular matrix is important for fabricating three‐dimensional (3D) scaffolds for tissue regeneration. A collagen‐like polypeptide, poly(Pro‐Hyp‐Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro‐Hyp‐Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible‐light photocrosslinking at various concentrations of methacrylated poly(Pro‐Hyp‐Gly) (20–50 mg/ml) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived 7 days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with scanning electron microscope observation, Alizarin Red S, and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/ml of methacrylated poly(Pro‐Hyp‐Gly) with 5‐min irradiation. These findings demonstrate that poly(Pro‐Hyp‐Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro‐Hyp‐Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration.     

Bioceramic akermanite enhanced vascularization and osteogenic differentiation of human induced pluripotent stem cells in 3D scaffolds in vitro and vivo

A growing number of studies suggest that the modulation of cell differentiation by biomaterials is critical for tissue engineering. In previous work, we demonstrated that human induced pluripotent stem cells (iPSCs) are remarkably promising seed cells for bone tissue engineering. In addition, we found that the ionic products of akermanite (Aker) are potential inducers of osteogenic differentiation of iPSCs. Furthermore, composite scaffolds containing polymer and bioceramics have more interesting properties compared to pure bioceramic scaffolds for bone tissue engineering. The characteristic of model biomaterials in bone tissue engineering is their ability to control the osteogenic differentiation of stem cells and simultaneously induce the angiogenesis of endothelia cells. Thus, this study aimed at investigating the effects of poly(lactic-co-glycolic acid)/Aker (PLGA-Aker) composite scaffolds on angiogenic and osteogenic differentiation of human iPSCs in order to optimize the scaffold compositions. The results from Alizarin Red S staining, qRT-PCR analysis of osteogenic genes (BMP2, RUNX2, ALP, COL1 and OCN) and angiogenic genes (VEGF and CD31) demonstrated that PLGA/Aker composite scaffolds containing 10% Aker exhibited the highest stimulatory effects on the osteogenic and angiogenic differentiation of human iPSCs among all scaffolds. After the scaffolds were implanted in nu/nu mice subcutaneous pockets and calvarial defects, H&E staining, BSP immunostaining, qRT-PCR analysis and micro-CT analysis (BMD, BV/TV) indicated that PLGA + 10% Aker scaffolds enhanced the vascularization and osteogenic differentiation of human iPSCs and stimulated the repair of bone defects. Taken together, our work indicated that combining scaffolds containing silicate bioceramic Aker and human iPSCs is a promising approach for the enhancement of bone regeneration.

Bioceramics akermanite enhanced vascularization and osteogenic differentiation of human iPSCs in 3D scaffolds in vitro and vivo.     

RGD‐functionalized polyurethane scaffolds promote umbilical cord blood mesenchymal stem cell expansion and osteogenic differentiation

Biomimetic materials are essential for the production of clinically relevant bone grafts for bone tissue engineering applications. Their ability to modulate stem cell proliferation and differentiation can be used to harness the regenerative potential of those cells and optimize the efficiency of engineered bone grafts. The arginyl‐glycyl‐aspartic acid (RGD) peptide has been recognized as the adhesion motif of various extracellular matrix proteins and can affect stem cell behaviour in biomaterials. Attempts to functionalize biomaterials with RGD have been limited to a maximum of 1‐ to 3‐mm thickness scaffolds, overlooking the issue of core infiltration that represents a major hurdle in developing real thickness scaffolds. Herein, we present the cross‐linking of RGD on the surface of “real thickness” (5 × 5 × 5 mm) porous polyurethane scaffolds (PU‐RGD), to be used for the expansion and osteogenic differentiation of umbilical cord blood mesenchymal stem cells (UCB MSCs). RGD‐functionalized scaffolds increased initial cell adhesion (1.5‐fold to twofold) and achieved a 3.4‐fold increase in cell numbers at the end of culture compared with a 1.5‐fold increase in non‐functionalized controls. Homogenous cell infiltration to the scaffold core was observed in the PU‐RGD scaffolds. Importantly, PU‐RGD scaffolds were able to enhance the osteogenic differentiation of UCB MSCs. Osteogenic gene and protein expression increased in scaffolds functionalized with 100 μg/ml RGD. Higher RGD concentrations (200 μg/ml) were less efficient in stimulating osteogenic differentiation. We conclude that robust RGD tethering to 3D PU “real thickness” scaffolds is possible and that it promotes core infiltration, expansion, and osteogenic differentiation of UCB MSCs for the purposes of bone regeneration.     

Synergistic effect of scaffold composition and dynamic culturing environment in multilayered systems for bone tissue engineering

Bone extracellular matrix (ECM) is composed of mineralized collagen fibrils which support biological apatite nucleation that participates in bone outstanding properties. Understanding and mimicking bone morphological and physiological parameters at a biological scale is a major challenge in tissue engineering scaffolding. Using emergent (nano)technologies scaffold designing may be critically improved, enabling highly functional tissue substitutes for bone applications. This study aims to develop novel biodegradable composite scaffolds of tricalcium phosphate (TCPs) and electrospun nanofibers of poly(?‐caprolactone) (PCL), combining TCPs osteoconductivity with PCL biocompatibility and elasticity, mimicking bone structure and composition. We hypothesized that scaffolds with such structure/composition would stimulate the proliferation and differentiation of bone marrow stromal cells (BMSCs) towards the osteogenic phenotype. Composite scaffolds, developed by electrospining using consecutive stacked layers of PCL and TCPs, were characterized by FTIR spectroscopy, X‐Ray diffraction and scanning electronic microscopy. Cellular behavior was assessed in goat BMSCs seeded onto composite scaffolds and cultured in static or dynamic conditions, using basal or osteogenic media during 7, 14 or 21 days. Cellular proliferation was quantified and osteogenic differentiation confirmed by alkaline phosphatase activity, alizarin red staining and immunocytochemistry for osteocalcin and collagen I. Results suggest that PCL‐TCP scaffolds provide a 3D support for gBMSCs proliferation and osteogenic differentiation with production of ECM. TCPs positively stimulate the osteogenic process, especially under dynamic conditions, where PCL‐TCP scaffolds are sufficient to promote osteogenic differentiation even in basal medium conditions. The enhancement of the osteogenic potential in dynamic conditions evidences the synergistic effect of scaffold composition and dynamic stimulation in gBMSCs osteogenic differentiation. Copyright © 2012 John Wiley & Sons, Ltd.     

RGD‐mimic polyamidoamine–montmorillonite composites with tunable stiffness as scaffolds for bone tissue‐engineering applications

This paper reports on the development of montmorillonite (MMT)‐reinforced hydrogels, based on a peptidomimetic polyamidoamine carrying guanidine pendants (AGMA1), as substrates for the osteo‐induction of osteoblast precursor cells. AGMA1 hydrogels of various degrees of crosslinking responded favourably to MMT reinforcement, giving rise to composite hydrogels with shear storage modulus G′, when fully swollen in water, up to 200 kPa, i.e. 20 times higher than the virgin hydrogels and of the same order or higher than other hydrogel‐based composites proposed for orthopaedic applications. This significant improvement was ascribed to the effective interpenetration between the polymer matrix and the inorganic filler. AGMA1–MMT hydrogels, when evaluated as scaffolds for the osteogenic differentiation of mouse calvaria‐derived pre‐osteoblastic MC3T3‐E1 cells, proved able to support cell adhesion and proliferation and clearly induced differentiation towards the osteoblastic phenotype, as indicated by different markers. In addition, AGMA1–MMT hydrogels proved completely degradable in aqueous media at pH 7.4 and did not provide any evidence of cytotoxicity. The experimental evidence suggests that AGMA1–MMT composites definitely warrant potential as scaffolds for osteoblast culture and bone grafts. Copyright © 2016 John Wiley & Sons, Ltd.