Enhanced Cells Anchoring to Electrospun Hybrid Scaffolds With PHBV and HA Particles for Bone Tissue Regeneration.
ABSTRACT: Hybrid materials combining organic and inorganic compounds used as scaffolds are highly beneficial in bone regeneration. In this study, we successfully produced by blend electrospinning poly(3-hydroxybutyric acid-co-3-hydrovaleric acid) (PHBV) scaffolds enriched with hydroxyapatite (HA) particles to biomimic bone tissue for improved and faster regeneration processes. The morphology, fiber diameters, and composition of the scaffolds were investigated by scanning electron microscopy (SEM) techniques followed by focused ion beam (FIB) sectioning to verify HA particles integration with PHBV fibers. In vitro cell culture was performed for 7 days and followed with the cell proliferation test (CellTiter-Blue® Assay). Additionally, cell integration with the scaffold was visualized by confocal and SEM imaging. We developed a simple way of obtaining hybrid scaffolds by electrospinning PHBV solution with HA particles without any post-processing. The PHBV + HA scaffold enhanced cell proliferation and filopodia formation responsible for cell anchoring within the created 3D environment. The obtained results show the great potential in the development of hybrid scaffolds stimulating bone tissue regeneration.
Project description:Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm(-1) can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.
Project description:Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and cellulose acetate (CA) were blended in the presence of a plasticizer, i.e., triethyl citrate (TEC), and a chain extender, i.e., poly(styrene-acrylic-co-glycidyl methacrylate). To increase the ductility and impact properties of PHBV and to investigate a new biodegradable PHBV-based blend for sustainable packaging, CA was compatibilized with TEC. PHBV and plasticized CA (pCA) blends showed complete immiscibility through separate glass transition and melting peak temperatures in differential scanning calorimetry (DSC), despite the similar Hansen solubility parameters of PHBV, CA, and TEC, indicating partial miscibility. Phase separation between PHBV and pCA was clearly observed by scanning electron microscopy (SEM). PHBV/pCA (70:30) blends had improved impact strength, exceeding that of neat PHBV and pCA, which is attributed to PHBV porosity induced by degradation from the high processing temperature. During processing, the plasticizer migrated from CA to PHBV and partially plasticized it, as evidenced through DSC analysis. The melt temperature of PHBV was reduced, which was confirmed by double melting peaks, representing the formation of secondary crystallites at a lower temperature. Due to processing at high temperatures (210-220 °C), significant porosity was observed in the PHBV/pCA 30:70 blend in SEM analysis. Consequently, the impact strength was improved by 110% as compared to that of virgin PHBV. The addition of CE had no effect on the mechanical properties but did make the PHBV/pCA blends morphologically uniform.
Project description:To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40?µm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray µCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10?day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue.
Project description:Dental implant surgeries involve the insertion of implant fixtures into alveolar bones to replace missing teeth. When the availability of alveolar bone at the surgical site is insufficient, bone graft particles are filled in the insertion site for successful bone reconstruction. Bone graft particles induce bone regeneration over several months at the insertion site. Subsequently, implant fixtures can be inserted at the recipient site. Thus, conventional dental implant surgery is performed in several steps, which in turn increases the treatment period and cost involved. Therefore, to reduce surgical time and minimize treatment costs, a novel hybrid scaffold filled with bone graft particles that could be combined with implant fixtures is proposed. This scaffold is composed of a three-dimensionally (3D) printed polycaprolactone (PCL) frame and osteoconductive ceramic materials such as hydroxyapatite (HA) and ?-tricalcium phosphate (?-TCP). Herein, we analyzed the porosity, internal microstructure, and hydrophilicity of the hybrid scaffold. Additionally, Saos-2 cells were used to assess cell viability and proliferation. Two types of control scaffolds were used (a 3D printed PCL frame and a hybrid scaffold without HA/?-TCP particles) for comparison, and the fabricated hybrid scaffold was verified to retain osteoconductive ceramic particles without losses. Moreover, the fabricated hybrid scaffold had high porosity and excellent microstructural interconnectivity. The in vitro Saos-2 cell experiments revealed superior cell proliferation and alkaline phosphatase assay results for the hybrid scaffold than the control scaffold. Hence, the proposed hybrid scaffold is a promising candidate for minimizing cost and duration of dental implant surgery.
Project description:Eugenyl acetate obtained via enzymatic esterification using Lipozyme TL IM enzyme was encapsulated in biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) through solution-enhanced dispersion by supercritical fluids (SEDS). Produced particles were characterized by SEM and confocal microscopy techniques and in addition in vitro release assays were performed in isopropanol and ethyl acetate. Experimental micronization conditions comprised 8 and 10 MPa, 308 and 313 K and eugenyl acetate concentration ranging from 5 to 20 mg mL-1, keeping PHBV concentration constant (20 mg mL-1 in dichloromethane). The maximum encapsulation efficiency was 58.0 % for 5 mg mL-1of eugenyl acetate at 8 MPa and 308 K. The morphology of the encapsulated particles for most of the trials was spherical, with particle size ranging from 0.061 to 0.276 ?m. Regarding the release in ethyl acetate and isopropanol solvents the higher the affinity of the encapsulated ester of these solvents, the faster the release was observed. These results demonstrate the importance of essential clove oil esterification reaction and encapsulation of the ester by SEDS method so that this encapsulated ester can be used in different industrial applications.
Project description:Tissue-engineered approaches to regenerate bone in the craniomaxillofacial region utilize biomaterial scaffolds to provide structural and biological cues to stem cells to stimulate osteogenic differentiation. Bioactive scaffolds are typically comprised of natural components but often lack the manufacturability of synthetic materials. To circumvent this trade-off, we 3D printed materials comprised of decellularized bone (DCB) matrix particles combined with polycaprolactone (PCL) to create novel hybrid DCB:PCL scaffolds for bone regeneration. Hybrid scaffolds were readily printable at compositions of up to 70% bone by mass and displayed robust mechanical properties. Assessments of surface features revealed both collagenous and mineral components of bone were present. Qualitative and quantitative assessments showed increased surface roughness relative to that of pure PCL scaffolds. These findings correlated with enhanced cell adhesion on hybrid surfaces relative to that on pure surfaces. Human adipose-derived stem cells (hASCs) cultured in DCB:PCL scaffolds without soluble osteogenic cues exhibited significant upregulation of osteogenic genes in hybrid scaffolds relative to pure PCL scaffolds. In the presence of soluble phosphate, hybrid scaffolds resulted in increased calcification. The hASC-seeded scaffolds were implanted into critical-sized murine calvarial defects and yielded greater bone regeneration in DCB:PCL scaffolds compared to that in PCL-only at 1 and 3 months post-transplantation. Taken together, these results demonstrate that 3D printed DCB:PCL scaffolds might be effective for stimulating bone regeneration.
Project description:BACKGROUND:Bone marrow-derived stem cells (BMSCs) and chondrocytes have been reported to present "dedifferentiation" and "phenotypic loss" during the chondrogenic differentiation process in cartilage tissue engineering, and cartilage progenitor cells (CPCs) are novel seeding cells for cartilage tissue engineering. In our previous study, cartilage progenitor cells from different subtypes of cartilage tissue were isolated and identified in vitro, but the study on in vivo chondrogenic characteristics of cartilage progenitor cells remained rarely. In the current study, we explored the feasibility of combining cartilage progenitor cells with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics of cartilage progenitor cells with those of bone marrow-derived stem cells and chondrocytes. METHODS:These three cells combined with PHBV were cultured in vitro for 1 week without chondrogenic induction and then transplanted subcutaneously into nude mice for 6 weeks. The cell-PHBV constructs were evaluated by gross observation, histological staining, glycosaminoglycan content measurement, biomechanical analysis and RT-PCR. RESULTS:The chondrocyte-PHBV constructs and CPC-PHBV constructs became an ivory-whitish cartilage-like tissue, while the BMSC-PHBV constructs became vascularized 6 weeks after the subcutaneous implantation. Histological examination showed that many typical cartilage structures were present in the chondrocyte group, some typical cartilage structures were observed in the CPC group, while no typical cartilage structures were observed in the BMSC group. CONCLUSIONS:Cartilage progenitor cells may undergo chondrogenesis without chondrogenic induction and are better at chondrogenesis than BMSCs but worse than chondrocytes in the application of cartilage tissue engineering.
Project description:The regeneration of functional tissue in osseous defects is a formidable challenge in orthopedic surgery. In the present study, a novel biomimetic composite scaffold, here called nano-hydroxyapatite (HA)/poly-?-caprolactone (PCL) was fabricated using a selective laser sintering technique. The macrostructure, morphology, and mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the nano-HA/PCL scaffolds exhibited predesigned, well-ordered macropores and interconnected micropores. The scaffolds have a range of porosity from 78.54% to 70.31%, and a corresponding compressive strength of 1.38 MPa to 3.17 MPa. Human bone marrow stromal cells were seeded onto the nano-HA/PCL or PCL scaffolds and cultured for 28 days in vitro. As indicated by the level of cell attachment and proliferation, the nano-HA/PCL showed excellent biocompatibility, comparable to that of PCL scaffolds. The hydrophilicity, mineralization, alkaline phosphatase activity, and Alizarin Red S staining indicated that the nano-HA/PCL scaffolds are more bioactive than the PCL scaffolds in vitro. Measurements of recombinant human bone morphogenetic protein-2 (rhBMP-2) release kinetics showed that after nano-HA was added, the material increased the rate of rhBMP-2 release. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both nano-HA/PCL scaffolds and PCL scaffolds were implanted in rabbit femur defects for 3, 6, and 9 weeks. The wounds were studied radiographically and histologically. The in vivo results showed that both nano-HA/PCL composite scaffolds and PCL scaffolds exhibited good biocompatibility. However, the nano-HA/PCL scaffolds enhanced the efficiency of new bone formation more than PCL scaffolds and fulfilled all the basic requirements of bone tissue engineering scaffolds. Thus, they show large potential for use in orthopedic and reconstructive surgery.
Project description:To regenerate the bone tissue, the fabrication of scaffolds for better tissue regeneration has attracted a great deal of attention. In fact, growth factors are already used in clinical practice and are being investigated for enhancing the capacity for bone tissue regeneration. However, despite their strong osteoinductive activity, these growth factors have several limitations: safety issues, high treatment costs, and the potential for ectopic bone formation. The aim of this study was therefore to develop ceramic scaffolds that could promote the capacity for bone regeneration without growth factors. Three-dimensional ceramic scaffolds were successfully fabricated from hydroxyapatite (HA) and tricalcium phosphate (TCP) using projection-based microstereolithography, which is an additive manufacturing technology. The effects of calcium ions released from ceramic scaffolds on osteogenic differentiation and bone regeneration were evaluated in vitro and in vivo. The osteogenesis-related gene expression and area of new bone formation in the HA/TCP scaffolds was higher than those in the HA scaffolds. Moreover, regenerated bone tissue in HA/TCP scaffolds were more matured than that in HA scaffolds. Through this study, we were able to enhance the bone regeneration capacity of scaffolds not by growth factors but by calcium ions released from the scaffolds. Ceramic scaffolds developed in this study might be useful for enhancing the capacity for regeneration in complex bone defects.
Project description:Glow discharge plasma (GDP) treatments of biomaterials, such as hydroxyapatite/?-tricalcium phosphate (HA/?-TCP) composites, produce surfaces with fewer contaminants and may facilitate cell attachment and enhance bone regeneration. Thus, in this study we used argon glow discharge plasma (Ar-GDP) treatments to modify HA/?-TCP particle surfaces and investigated the physical and chemical properties of the resulting particles (HA/?-TCP + Ar-GDP). The HA/?-TCP particles were treated with GDP for 15 min in argon gas at room temperature under the following conditions: power: 80 W; frequency: 13.56 MHz; pressure: 100 mTorr. Scanning electron microscope (SEM) observations showed similar rough surfaces of HA/?-TCP + Ar-GDP HA/?-TCP particles, and energy dispersive spectrometry analyses showed that HA/?-TCP surfaces had more contaminants than HA/?-TCP + Ar-GDP surfaces. Ca/P mole ratios in HA/?-TCP and HA/?-TCP + Ar-GDP were 1.34 and 1.58, respectively. Both biomaterials presented maximal intensities of X-ray diffraction patterns at 27° with 600 a.u. At 25° and 40°, HA/?-TCP + Ar-GDP and HA/?-TCP particles had peaks of 200 a.u., which are similar to XRD intensities of human bone. In subsequent comparisons, MG-63 cell viability and differentiation into osteoblast-like cells were assessed on HA/?-TCP and HA/?-TCP + Ar-GDP surfaces, and Ar-GDP treatments led to improved cell growth and alkaline phosphatase activities. The present data indicate that GDP surface treatment modified HA/?-TCP surfaces by eliminating contaminants, and the resulting graft material enhanced bone regeneration.