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Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. Here, monodisperse templated scaffolds (MTSC) were fabricated and used as a well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress (WSS) using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cells differentiation, bone-like tissue formation, structure and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and micro-computed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared to static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, while MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared to MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs.Impact statementAging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction towards the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

Our goal was to create bioimitated scaffolding materials for biomedical purposes. The guiding idea was that we used an interpenetrating structural hierarchy of natural extracellular matrix as a “pattern” to design hydrogel scaffolds that show favorable properties for tissue regeneration. Polymeric hydrogel scaffolds are made in a simple, environmentally friendly way without additional functionalization. Gelatin and 2-hydroxyethyl methacrylate were selected to prepare interpenetrating polymeric networks and linear alginate chains were added as an interpenetrant to study their influence on the scaffold’s functionalities. Cryogelation and porogenation methods were used to obtain the designed scaffolding biomaterials. The scaffold’s structural, morphological, and mechanical properties, in vitro degradation, and cell viability properties were assessed to study the effects of the preparation method and alginate loading. Apatite as an inorganic agent was incorporated into cryogelated scaffolds to perform an extensive biological assay. Cryogelated scaffolds possess superior functionalities essential for tissue regeneration: fully hydrophilicity, degradability and mechanical features (2.08–9.75 MPa), and an optimal LDH activity. Furthermore, cryogelated scaffolds loaded with apatite showed good cell adhesion capacity, biocompatibility, and non-toxic behavior. All scaffolds performed equally in terms of metabolic activity and osteoconductivity. Cryogelated scaffolds with/without HAp could represent a new advance to promote osteoconductivity and enhance hard tissue repair. The obtained series of scaffolding biomaterials described here can provide a wide range of potential applications in the area of biomedical engineering.

Our goal was to create bioimitated scaffolding materials for biomedical purposes. The guiding idea was that we used an interpenetrating structural hierarchy of natural extracellular matrix as a "pattern" to design hydrogel scaffolds that show favorable properties for tissue regeneration. Polymeric hydrogel scaffolds are made in a simple, environmentally friendly way without additional functionalization. Gelatin and 2-hydroxyethyl methacrylate were selected to prepare interpenetrating polymeric networks and linear alginate chains were added as an interpenetrant to study their influence on the scaffold's functionalities. Cryogelation and porogenation methods were used to obtain the designed scaffolding biomaterials. The scaffold's structural, morphological, and mechanical properties, in vitro degradation, and cell viability properties were assessed to study the effects of the preparation method and alginate loading. Apatite as an inorganic agent was incorporated into cryogelated scaffolds to perform an extensive biological assay. Cryogelated scaffolds possess superior functionalities essential for tissue regeneration: fully hydrophilicity, degradability and mechanical features (2.08-9.75 MPa), and an optimal LDH activity. Furthermore, cryogelated scaffolds loaded with apatite showed good cell adhesion capacity, biocompatibility, and non-toxic behavior. All scaffolds performed equally in terms of metabolic activity and osteoconductivity. Cryogelated scaffolds with/without HAp could represent a new advance to promote osteoconductivity and enhance hard tissue repair. The obtained series of scaffolding biomaterials described here can provide a wide range of potential applications in the area of biomedical engineering.

Mechanical loading has been shown to influence various osteogenic responses of bone-derived cells and bone formation in vivo. However, the influence of mechanical stimulation on the formation of bone organoid in vitro is not clearly understood. Here, three-dimensional (3D) bioprinted human mesenchymal stem cells-laden graphene oxide composite scaffolds were cultured in a novel cyclic-loading bioreactors for up to 56 d. Our results showed that mechanical loading from day 1 (ML01) significantly increased organoid mineral density, organoid stiffness, and osteoblast differentiation compared with non-loading and mechanical loading from day 21. Importantly, ML01 stimulated collagen I maturation, osteocyte differentiation, lacunar-canalicular network formation and YAP expression on day 56. These finding are the first to reveal that long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids. Such 3D bone organoids may serve as a human-specific alternative to animal testing for the study of bone pathophysiology and drug screening., Biofabrication, 14 (3), ISSN:1758-5082, ISSN:1758-5090

Mechanical loading has been shown to influence various osteogenic responses of bone-derived cells and bone formation in vivo. However, the influence of mechanical stimulation on the formation of bone organoid in vitro is not clearly understood. Here, 3D bioprinted human mesenchymal stem cells (hMSCs)-laden graphene oxide composite scaffolds were cultured in cyclic-loading bioreactors for up to 56 days. Our results showed that mechanical loading from day 1 (ML01) significantly increased organoid mineral density, organoid stiffness, and osteoblast differentiation compared with non-loading and mechanical loading from day 21. Importantly, ML01 stimulated collagen I maturation, osteocyte differentiation, lacunar-canalicular network formation and YAP expression on day 56. These finding are the first to reveal that long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids. Such 3D bone organoids may serve as a human-specific alternative to animal testing for the study of bone pathophysiology and drug screening.

New composite 3D scaffolds were developed as a combination of synthetic polymer, poly(2-hydroxyethyl methacrylate) (PHEMA), and a natural polymer, gelatin, with a ceramic component, nanohydroxyapatite (ID nHAp) dopped with metal ions. The combination of a synthetic polymer, to be able to tune the structure and the physicochemical and mechanical properties, and a natural polymer, to ensure the specific biological functions of the scaffold, with inorganic filler was applied. The goal was to make a new material with superior properties for applications in the biomedical field which mimics as closely as possible the native bone extracellular matrix (ECM). Biodegradable PHEMA hydrogel was obtained by crosslinking HEMA by poly(β-amino esters) (PBAE). The scaffold’s physicochemical and mechanical properties, in vitro degradation, and biological activity were assessed so to study the effects of the incorporation of nHAp in the (PHEMA/PBAE/gelatin) hydrogel, as well as the effect of the different pore-forming methods. Cryogels had higher elasticity, swelling, porosity, and percent of mass loss during degradation than the samples obtained by porogenation. The composite scaffolds had a higher mechanical strength, 10.14 MPa for the porogenated samples and 5.87 MPa for the cryogels, but a slightly lower degree of swelling, percent of mass loss, and porosity than the hybrid ones. All the scaffolds were nontoxic and had a high cell adhesion rate, which was 15–20% higher in the composite samples. Cell metabolic activity after 2 and 7 days of culture was higher in the composites, although not statistically different. After 28 days, cell metabolic activity was similar in all scaffolds and the TCP control. No effect of integrating nHAp into the scaffolds on osteogenic cell differentiation could be observed. Synergetic effects occurred which influenced the mechanical behavior, structure, physicochemical properties, and interactions with biological species.

Bioprinting is a promising technique for facilitating the fabrication of engineered bone tissues for patient-specific defect repair and for developing in vitro tissue/organ models for ex vivo tests. However, polymer-based ink materials often result in insufficient mechanical strength, low scaffold fidelity and loss of osteogenesis induction because of the intrinsic swelling/shrinking and bioinert properties of most polymeric hydrogels. Here, we developed a human mesenchymal stem cells (hMSCs)-laden graphene oxide (GO)/alginate/gelatin composite bioink to form 3D bone-mimicking scaffolds using a 3D bioprinting technique. Our results showed that the GO composite bioinks (0.5GO, 1GO, 2GO) with higher GO concentrations (0.5, 1 and 2 mg/ml) improved the bioprintability, scaffold fidelity, compressive modulus and cell viability at day 1. The higher GO concentration increased the cell body size and DNA content, but the 2GO group swelled and had the lowest compressive modulus at day 42. The 1GO group had the highest osteogenic differentiation of hMSC with the upregulation of osteogenic-related gene (ALPL, BGLAP, PHEX) expression. To mimic critical-sized calvarial bone defects in mice and prove scaffold fidelity, 3D cell-laden GO defect scaffolds with complex geometries were successfully bioprinted. 1GO maintained the best scaffold fidelity and had the highest mineral volume after culturing in the bioreactor for 42 days. In conclusion, GO composite bioinks had better bioprintability, scaffold fidelity, cell proliferation, osteogenic differentiation and ECM mineralization than the pure alginate/gelatin system. The optimal GO group was 1GO, which demonstrated the potential for 3D bioprinting of bone tissue models and tissue engineering applications., Acta Biomaterialia, 121, ISSN:1742-7061, ISSN:1878-7568

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteogenic cellular organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs. 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteoblast- and early osteocyte-related gene expression and 3D cellular network within the mineralized matrix at day 42. Furthermore, the results showed that cell density in 15 M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5 M cells/ml had the significantly higher mineral formation rate than 15 M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications. Statement of Significance: In this study, we presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-CT monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density. The soft scaffolds had higher cell proliferation, enhanced alkaline phosphatase activity and stimulated osteogenic differentiation with 3D cellular network foramtion than stiff scaffolds. Significantly more mineralized bone-like tissue was formed in soft scaffolds than stiff scaffolds at day 42. Meanwhile, cell density in 15 M cells/ml can promote cell-cell connections and mineral formation in 14 days, while the higher mineral formation rate was found in 5 M cells/ml from day 14 to day 21. © 2020 Acta Materialia Inc., Acta Biomaterialia, 114, ISSN:1742-7061, ISSN:1878-7568

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteoblast- and osteocyte-like cell organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified FDA-compliant polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteocyte-related gene expression at day 42. Furthermore, the results showed that cell density in 15M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5M cells/ml had the significantly higher mineral formation rate than 15M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications.

The poor innate healing capacity of fibroblastic tissues, such as pelvic floor fascia, is attributed to the scarcity of fibroblasts to produce collagen, as the main collagen producing cells. Coaxial electrospun PCL/PEO fibers containing basic fibroblast growth factor (FGF-2) were evaluated for the local and temporal delivery of FGF-2 for promoting fibroblast proliferation. PCL/PEO coaxial fibers with a highly porous surface were successfully developed using coaxial electrospinning. The diameter of the PCL/PEO microfibers produced by coaxial electrospinning could be tuned by the electrospinning parameters. XPS surface chemistry probing and CA wettability analysis confirmed that the outer surface of the coaxial fibers is PCL. The protein was successfully encapsulated and a sustained release was observed over more than 9 days. In vitro, PCL/PEO coaxial fibers supported fibroblast cell adhesion. In addition, PCL/PEO coaxial fibers containing FGF-2 significantly enhanced fibroblast cell viability and proliferation. Further, Coll-I expression was significantly expressed after day 1 while down-regulated after day 9 compared to the control group. These results indicate that coaxial polymeric fibers allow local and sustained growth factor delivery with prolonged efficacy and longevity for connective tissue regeneration.

Three-dimensional (3D) cell-laden scaffolds are becoming more prevalent in bone tissue repair and regeneration. However, the influence of physical scaffold properties on cell behavior is still unclear. In this study, we fabricated four different alginate concentration (0.8, 1.3, 1.8 and 2.3%alg) composite cell-laden porous scaffolds using a 3D bioprinting technique. The aim was to investigate the changes of physical properties affected by the alginate concentration and the influences on cell behavior. The study showed that the different alginate concentration scaffolds had uniform macropores (500-600 μm) with compressive moduli ranging from 1.5 kPa (0.8%alg) to 14.2 kPa (2.3%alg). Long-term structural integrity of the printed scaffolds was achieved when cultured in cell culture media, but not when cultured in phosphate buffered saline (PBS). Scaffold structure, swelling behavior, and compressive moduli decreased with culturing time and higher alginate concentration lead to more stable physical scaffold properties. Meanwhile, human mesenchymal stem cell (hMSCs) laden non-printed and bioprinted composite scaffolds were fabricated. Bioprinting did not affect cell viability, but alginate concentration had a significant influence on cell viability and cell morphology. Lower alginate concentration scaffolds (0.8%alg) showed higher cell viability (84% ± 0.7% versus 68% ± 1.3%) compared to higher alginate concentration scaffolds (2.3%alg) at day 14. Live cell image in the 0.8%alg scaffolds demonstrated the formation of a 3D interconnected cellular network, while cells in the 1.8 and 2.3%alg scaffolds formed spheroids. In conclusion, this study broadens the design space for alginate-based bioinks for 3D bioprinting. Higher alginate concentration preserved better scaffold fidelity and mechanical properties. Better cell viability and cell spreading morphology was achieved in lower alginate concentration scaffolds, which is relevant for potential applications in bone tissue engineering.

PURPOSE Previous studies have demonstrated the capacity of a designed proline-rich synthetic peptide to stimulate osteoblast differentiation and biomineralization in vitro. Therefore, the aim of the present study was to evaluate the osseointegration capacity of titanium (Ti) implants coated with these peptides in a rabbit model. MATERIALS AND METHODS Four calibrated defects were prepared in the tibiae of three New Zealand rabbits, and the defects were randomized into a test group (peptide-modified machined Ti implant) and a control group (unmodified machined Ti implant). The performance in vivo was investigated after 4 weeks of implantation by real-time reverse transcriptase polymerase chain reaction of bone and inflammatory markers, microcomputed tomographic analysis of mineralized bone, and histologic examination. RESULTS The peptides adsorbed in agglomerates on Ti and underwent a change in secondary structure upon adsorption, which induced an increase in surface wettability. Gene expression markers indicated that peptide-coated Ti implants had significantly decreased mRNA levels of tartrate-resistant acid phosphatase. A trend toward increased osteocalcin in the peri-implant bone tissue was also seen. Bone morphometric and histologic parameters did not show significant differences, although the peptide group showed a higher percentage of new bone histologically. CONCLUSIONS Proline-rich peptides have potential as a biocompatible coating for promoting osseointegration of Ti implants by reducing bone resorption.

The aim of this study was to investigate the effect of TiO2 scaffold (SC) coated with an alginate hydrogel containing a proline-rich peptide (P2) on osteoblast proliferation and differentiation in vitro. Peptide release was evaluated and a burst release was observed during the first hours of incubation, and then progressively released overtime. No changes were observed in the cytotoxicity after 48 h of seeding MC3T3-E1 cells on the coated and uncoated TiO2 SC. The amount of cells after 7 days was higher on uncoated TiO2 SC than on alginate-coated TiO2 SC, measured by DNA content and scanning electron microscope imaging. In addition, while lower expression of integrin beta1 was detected for alginate-coated TiO2 SC at this time point, similar gene expression was observed for other integrins, fibronectin-1, and several osteoblast differentiation markers. After 21 days, gene expression of integrin beta3, fibronectin-1, osterix, and collagen-I was increased in alginate-coated compared to TiO2 SC. Moreover, increased gene expression of integrin alpha8, bone morphogenetic protein 2, interleukin-6, and collagen-I was found on P2 alginate-coated TiO2 SC compared to alginate-coated TiO2 SC. In conclusion, our results indicate that alginate-coated TiO2 SC can act as a matrix for delivery of proline-rich peptides increasing osteoblast differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Proton pump inhibitors (PPIs) are widely used against gastroesophageal reflux disease. Recent epidemiological studies suggest that PPI users have an increased risk of fractures, but a causal relationship has been questioned. We have therefore investigated the skeletal phenotype in H(+) /K(+) ATPase beta-subunit knockout (KO) female mice. Skeletal parameters were determined in 6- and 20-month-old KO mice and in wild-type controls (WT). Whole body bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X-ray absorptiometry (DXA). Femurs were examined with µCT analyses and break force were examined by a three-point bending test. Plasma levels of gastrin, RANKL, OPG, osteocalcin, leptin, and PTH were analyzed. KO mice had lower whole body BMC at 6 months (0.53 vs. 0.59 g, P = 0.035) and at 20 months (0.49 vs. 0.74 g, P

Previous studies have shown that bone-to-implant attachment of titanium implants to cortical bone is improved when the surface is modified with hydrofluoric acid. The aim of this study was to investigate if biological factors are involved in the improved retention of these implants. Fluoride was implemented in implant surfaces by cathodic reduction with increasing concentrations of HF in the electrolyte. The modified implants were placed in the cortical bone in the tibias of New Zealand white rabbits. After 4 weeks of healing, wound fluid collected from the implant site showed lower lactate dehydrogenase activity and less bleeding in fluoride-modified implants compared to control. A significant increase in gene expression levels of osteocalcin and tartrate-resistant acid phosphatase (TRAP) was found in the cortical bone attached to Ti implants modified with 0.001 and 0.01 vol.% HF, while Ti implants modified with 0.1% HF showed only induced TRAP mRNA levels. These results were supported by the performed micro-CT analyses. The volumetric bone mineral density of the cortical bone hosting Ti implants modified with 0.001% and 0.01% HF was higher both in the newly woven bone (100 microm from the interface) and in the older Haversian bone (100 microm). In conclusion, the modulation of these biological factors by surface modification of titanium implants with low concentrations of HF using cathodic reduction may explain their improved osseointegration properties.

Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties including minimal metabolism, hepatic selectivity and enhanced inhibition of HMG-CoA reductase. An induction of osteoblast differentiation has been reported in vitro with lipophilic statins but not with RSV, which, like pravastatin, is relatively hydrophilic compared with other statins. To mediate its action, an active transport mechanism via solute carrier (SLC) transporters from the SLC16, SLC21/SLCO and SLC22 gene family - specifically Slc16a1, Slco1a1, Slco2b1 and Slc22a8 - may be present to allow effective entry in osteoblastic cells. In this study, we demonstrate that RSV induced osteoblast differentiation, as measured by increased BMP-2 gene expression and secretion, and ALP activity in MC3T3-E1 osteoblast cells, without significantly affecting cell proliferation within the concentration range of 0.001-10 μM. Low concentrations of RSV (0.001-0.01 μM) were protective against cell death whereas higher concentrations (10-100 μM) showed cytotoxicity. Moreover, MC3T3-E1 osteoblasts expressed high levels of Slco1a1 and Slc16a1 mRNA and low levels of Slco2b1 and Slc22a8 mRNA, when compared with kidney and liver tissues from mice. Slco1a1 gene expression increased 12-fold during osteoblast differentiation and was further regulated after RSV treatment. In conclusion, as for other statins, RSV promotes osteoblast differentiation, and also, demonstrated for the first time, regulates the expression of Slco1a1, which may constitute the transport system for RSV across the cell membrane in mature osteoblasts.

Fibroblasts are ubiquitous cells that constitute the stroma of virtually all tissues and play vital roles in homeostasis. The poor innate healing capacity of fibroblastic tissues is attributed to the scarcity of fibroblasts as collagen-producing cells. In this study, we have developed a functional ECM mimicking scaffold that is capable to supply spatial allocation of stem cells as well as anchorage and storage of growth factors (GFs) to direct stem cells differentiate towards fibroblasts. Electrospun PCL fibers were embedded in a PEG-fibrinogen (PF) hydrogel, which was infiltrated with connective tissue growth factor (CTGF) to form the 3D nanocomposite PFP-C. The human induced pluripotent stem cells derived mesenchymal stem cells (hiPS-MSCs) with an advance in growth over adult MSCs were applied to validate the fibrogenic capacity of the 3D nanocomposite scaffold. The PFP-C scaffold was found not only biocompatible with the hiPS-MSCs, but also presented intriguingly strong fibroblastic commitments, to an extent comparable to the positive control, tissue culture plastic surfaces (TCP) timely refreshed with 100% CTGF. The novel scaffold presented not only biomimetic ECM nanostructures for homing stem cells, but also sufficient cell-approachable bio-signaling cues, which may synergistically facilitate the control of stem cell fates for regenerative therapies.

Electrospinning of immiscible polymer blends of PCL and PEO has rendered the solid, straight and hydrophobic PCL fibers into porous, hydrophilic microfibers. In this study, electrospun PCL, 11.4%PEO–88.6%PCL and 23.1%PEO–76.9%PCL fibers were loaded with dexamethasone (DEX) without changing the morphology. Their anti-inflammatory properties on Raw 264.7 cells were compared in vitro. All fibers were found biocompatible and the encapsulation of DEX could alleviate the LPS induced inflammation response. Differences in surface topography, chemical composition, wettability and release kinetics among the different fibers collectively affected the regulation of inflammatory related gene expression.

Structured Abstract Objectives To investigate biological changes in alveolar bone occurring during orthodontic relapse. Materials and Methods Rat maxillary first molars were moved mesially for 10 days. After orthodontic tooth movement (OTM), appliances were removed, and the molars were allowed to relapse for one, three, five, seven, 14 or 21 days. Changes in 3D morphometric parameters of bone located mesial to the first molars were evaluated by micro-CT. Total RNA was isolated from the same bone site, and real-time RT-PCR was used to measure the expression of bone formation and resorption markers. Results One day after appliance removal, the molars relapsed to a mean 73% of the achieved OTM and then steadily relapsed to 93% at 21 days. Tissue mineral density and per cent bone volume increased over the experimental period. Inversely, there was a decrease in total porosity. Gene expression of OCN, Coll-I and ALP decreased during OTM, whilst as the molars relapsed showed tended to increase. Gene expression of RANKL and TRAP increased during OTM. Changes in mRNA expression of H+-ATPase were minor. By 21 days post-appliance removal, the remodelling process in rats appeared to have returned to control levels. Conclusions Bone tissue reactions on a molecular level are similar during OTM and orthodontic relapse. These findings validate the importance of immediate retention following active OTM.

One of the major challenges in the field of biomaterials is to develop strategies to regulate the innate host inflammatory response. Coaxial electrospun PCL/PEO hollow fibers containing dexamethasone were evaluated for a local delivery of dexamethasone to reduce adverse inflammation responses often resulting from biomaterial implantation. Core–shell PCL/PEO hollow fibers with a highly porous surface were successfully developed using coaxial electrospinning. The release of dexamethasone exhibited a burst release during the first 0.5–3 h followed by a sustained release over more than 12 days. In vitro study showed that the dexamethasone encapsulated coaxial PCL/PEO hollow fibers significantly reduced cell proliferation of LPS-stimulated macrophages and meanwhile significantly decreased the mRNA expression levels of the pro-inflammatory cytokines TNF-α and IL-1β. The dexamethasone encapsulated coaxial PCL/PEO hollow microfibers are promising biomaterials for implantation to combat the acute inflammation responses which take place during first 24–48 h after biomaterial implantation and meanwhile to regulate possible hostile chronic inflammations, thus increasing the efficacy and longevity of the implants in vivo.

The bone growth promoting effects of statins suggest that these bioactive molecules can be used to improve the integration of bone-anchored implants. This study aimed at the application of simvastatin with dental implants for use in patients with low bone density. Coin-shaped titanium zirconium samples with grit-blasted and acid-etched surface were coated with simvastatin, using a novel anodic oxidation setup under alkaline conditions. The presence of intact simvastatin attached to the surface was confirmed by infrared spectroscopy. A binding site on the aliphatic O-H group was discovered and the integration of 1H, 18O and 12C in the depth of the surface were observed by secondary ion mass spectroscopy. A simvastatin concentration of about 60 g/cm2 was found in a release study over 72 h. The simvastatin-coated surfaces promoted alkaline phosphatase, collagen type I and osteocalcin gene expression of MC3T3-E1 cells. This suggested that the demonstrated coating holds potential for use in patients with compromised bone.

The aim of this study was to show that cathodic polarization can be used for coating commercial implant surfaces with an immobilized but functional and bioavailable surface layer of strontium (Sr). Moreover, this study assessed the effect of fluorine on Sr-attachment. X-ray photoelectron spectroscopy revealed that addition of fluorine (F) to the buffer during coating increased surface Sr-amounts but also changed the chemical surface composition by adding SrF2 alongside of SrO whereas pre-treatment of the surface by pickling in hydrofluoric acid appeared to hinder Sr-attachment. Assessment of the bio-availability hinted at a positive effect of Sr on cell differentiation given that the surface reactivity of the original surface remained unchanged. Additional SrF2 on the surface appeared to reduce undesired surface contamination while maintaining the surface micro-topography and micro-morphology. Anyhow, this surface modification revealed to create nano-nodules on the surface.

Polyproline-rich synthetic peptides have previously been shown to induce bone formation and mineralization in vitro and to decrease bone resorption in vivo. Alginate hydrogel formulations containing these synthetic peptides (P2, P5, P6) or Emdogain® (EMD) were tested for surface coating of bone implants. In an aqueous environment, the alginate hydrogels disclosed a highly compact structure suitable for cell adhesion and proliferation. Lack of cytotoxicity of the alginate-gel coating containing peptides was tested in MC3T3-E1 cell cultures. In the present study, relative mRNA expression levels of integrin alpha 8 were induced by P5 compared to untreated alginate gel, and osteopontin mRNA levels were increased after 21 days of culture by treatment with synthetic peptides or EMD compared to control. Further, in agreement with previous results when the synthetic peptides were administered in the culture media, osteocalcin mRNA was significantly upregulated after long-term treatment with the formulated synthetic peptides compared to untreated and EMD alginate gel. These results indicate that the alginate gel is a suitable carrier for the delivery of synthetic peptides, and that the formulation is promising as biodegradable and biocompatible coating for bone implants.

Titanium oxide (TiO₂) scaffolds have previously been reported to exhibit very low mechanical strength. However, we have been able to produce a scaffold that features a high interconnectivity, a porosity of 91% and a compressive strength above 1.2 MPa. This study analyzed the in vivo performance of the porous TiO₂ scaffolds in a peri-implant cortical defect model in the rabbit. After 8 weeks of healing, morphological microcomputed tomography analyses of the defects treated with the TiO₂ scaffolds had significantly higher bone volume, bone surface and bone surface-to-volume ratio when compared to sham, both in the cortical and bone marrow compartment. No adverse effects, i.e. tissue necrosis or inflammation as measured by lactate dehydrogenase activity and real-time reverse transcription polymerase chain reaction analysis, were observed. Moreover, the scaffold did not hinder bone growth onto the adjacent cortical titanium implant. Histology clearly demonstrated new bone formation in the cortical sections of the defects and the presence of newly formed bone in close proximity to the scaffold surface and the surface of the adjacent Ti implant. Bone-to-material contact between the newly formed bone and the scaffold was observed in the histological sections. Islets of new bone were also present in the marrow compartment albeit in small amounts. In conclusion, the present investigation demonstrates that TiO₂ scaffolds osseointegrate well and are a suitable scaffold for peri-implant bone healing and growth.

With the aim of discovering new molecules for induction of bone formation and biomineralization, combination of bioinformatics and simulation methods were used to design the structure of artificial peptides based on proline-rich domains of enamel matrix proteins. In this study, the effect of such peptides on the differentiation toward the osteogenic lineage of human umbilical cord mesenchymal stem cells (hUCMSCs) was evaluated with or without osteogenic supplements (hydrocortisone, β-glycerol phosphate, and ascorbic acid) and compared to the effect of the commercially available enamel matrix derivative (EMD). It was hypothesized that the differentiation toward the osteogenic lineage of hUCMSCs would be promoted by the treatment with the synthetic peptides when combined with differentiation media, or it could even be directed exclusively by the synthetic peptides. Osteoinductivity was assessed by cell proliferation, bone morphogenetic protein-2 secretion, and gene expression of osteogenic markers after 1, 3, and 14 days of treatment. All peptides were safe with the dosages used, showing lower cell toxicity. P2, P4, and P6 reduced cell proliferation with growing media by 10%-15%. Higher expression of early osteoblast markers was found after 3 days of treatment with EMD in combination with osteogenic supplements, while after 14 days of treatment, cells treated by the different synthetic peptides in combination with osteogenic supplements showed higher osteocalcin mRNA levels. We can conclude that osteogenic differentiation of hUCMSCs is promoted by short-term EMD treatment in combination with osteogenic supplements and by long-term treatment by the synthetic peptides in combination with osteogenic supplements, showing similar results for all the peptide variants analyzed in this study.

The aim of this study was to design implant surfaces that attach less to bone but at the same time improve osseous healing for use as temporary bone fracture plates. The strategy was to combine the nonadhesive properties of smooth titanium (Ti) surfaces with the differentiative and anti-inflammatory properties of eicosapentaenoic acid (EPA). Machined Ti implant surfaces coated with a layer of EPA, with or without UV irradiation, were characterized by X-ray photoelectron spectroscopy, and their in vivo performance was evaluated in New Zealand White rabbits. The performance of the functionalised implants was analyzed after 10 weeks of healing by mechanical pull-out testing, molecular biology, and histological and microcomputed tomography analysis. The results indicate that surface functionalization with UV light can reduce bone attachment and volumetric bone mineral density in the peri-implant bone tissue. The presence of EPA on the surfaces enhanced this effect further. Gene expression of bone formation markers showed a trend toward higher mRNA levels in all EPA treated groups. The histological analyses demonstrated lower inflammation in the UV-irradiated group and immature bone formation in all the groups. In conclusion, surface functionalization of Ti implants with UV light and EPA could be a biocompatible coating for reduced bone bonding ability of Ti while promoting bone formation.

Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties, but its local effect in bone has yet to be investigated. The aim of this study was to evaluate the potential of absorbable collagen sponge (ACS) as a carrier for RSV to enhance bone formation in critical-size cortical bone defects adjacent to titanium implants. ACS, treated with different concentrations of RSV (R1 = 8.7 + or - 1.8 microg; R2 = 52.0 + or - 4.4 microg; R3 = 259.1 + or - 8.8 microg) or phosphate-buffered saline alone, were placed into the bone marrow through a defect made in the proximal tibial cortical bone of New Zealand White rabbits. One empty defect (SHAM) served as an internal control in each animal. After a healing time of 4 weeks, a concentration-dependent increase of alkaline phosphatase activity in ACS treated with RSV was detected in the bone fluid after removing the implants. In addition, a significant concentration-dependent increase in BMP-2 mRNA levels was found in the cortical bone tissue adjacent to the RSV-treated ACS. The cortical architecture of bone defects analysed by micro-computed tomography showed a trend towards higher bone volume in the ACS+R1 group compared with SHAM, which was accompanied by an increase in the bone mineral density. Evaluation of histological sections showed new bone formation in ACS treated with RSV but not in untreated ACS. These results indicate that RSV, when administered locally in bone, may have a potential effect in stimulating bone formation.

Fluoride-modification of dental titanium (Ti) implants is used to improve peri-implant bone growth and bone-to-implant contact and adhesion strength. In this study, the surface topography, chemistry and biocompatibility of polished Ti surfaces treated with hydrofluoric acid solution (HF) were studied. Murine osteoblasts (MC3T3-E1) were cultured on the different groups of Ti surfaces. Surfaces treated with HF had higher roughness, lower cytotoxicity level and better biocompatibility than controls. For short treatment times (40 and 90 s), fluorine was detected only within the first 5 nm of the surface layer (X-ray Photoemission Spectroscopy, XPS), whereas longer treatment time (120 and 150 s) caused fluoride ions to penetrate deeper (Secondary Ion Mass Spectrometry, SIMS). These results suggest that submerging Ti implants in a weak HF solution instigate time-dependant specific surface changes that are linked to the improved biocompatibility of these surfaces.

Production of one dimensional nanomaterials with secondary morphology exhibiting unique functions is challenging. Here we report for the first time that a nanoscale immiscible polymer blend solution electrojet can assemble into ultraporous interweaving microfibers. This intriguingly novel morphology originated from a blend of polycaprolactone (PCL) and polyethylene oxide (PEO) in a DCM-DMF mixed solution when the ratio between each component reached a threshold and when the electrospinning parameters were delicately controlled. The morphology, crystallinity, surface chemistry and wettabilities were characterized to understand the mechanism of formation. The interplay of the two semi-crystalline polymers and the pair of solvents/non-solvents with the electrospinning processing parameters was found to be critical for the formation of the unique structure. Furthermore, the interesting combination of biocompatible, biodegradable PCL with protein-resistant PEO motivated us to assess its inflammation responses on the RAW 264.7 macrophage cell line. All fibers were found to be biocompatible with low inflammation potential upon incubation, while compared with pure PCL nanofibers; the unique interweaving microfibers induced a slightly higher inflammatory reaction.

In this study, we compared the structural and physicochemical properties of different concentrations of alginate and high molecular weight hyaluronic acid (HA) hydrogels and their biocompatibility and bioactivity after long-term culture with MC3T3-E1 cells. Both hydrogels were biocompatible and supported long-term viability and cell proliferation. Alginate induced higher alkaline phosphatase (ALP) activity levels than HA. Calcium content was increased in concentration dependent manner in cells cultured with alginate compared to control. Culture with HA hydrogels reduced alkaline phosphatase (Alp), bone sialoprotein (Bsp) and osteocalcin (Oc), while alginate increased Oc mRNA levels. Unmodified alginate hydrogels supported osteoblast differentiation better than HA hydrogels, suggesting that alginates are more suitable for biomaterial applications in bone tissue engineering.

Eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) that has been reported to have beneficial effects on bone remodeling. Coating of titanium (Ti) implant surfaces with EPA could be used to achieve better bone healing. The aim of this study was to compare methods to coat polished Ti surfaces with a thin layer of EPA, either physically adsorbed or chemically bound, the latter being produced by UV irradiation of TiO2 and EPA. Surface characterization of the coatings was achieved by FTIR, profilometry, water contact angle (CA) measurements, and scintillation counting of [14C]-EPA. UV irradiation resulted in peroxidation of EPA molecules, while Ti surfaces became amphiphilic indicating dissociation of hydroxide molecules. The amount of [14C]-EPA that remained on UV irradiated surfaces was significantly higher than on non-irradiated surfaces. Surfaces with chemically bound EPA did not affect the viability of MC3T3-E1 cellsin vitro and were able to enhance cell attachment and differentiation, as indicated by increased gene expression of osteoblast-specific markers. Our results suggest that UV light is a suitable method to bind EPA on the surface of Ti disks, to improve cell attachment and differentiation of osteoblastic cells.