Sintered Microsphere Scaffolds Research Articles

Harnessing notch signaling to enhance osteoblast mineralization for scaffold-based bone regeneration

Event Abstract Back to Event Harnessing notch signaling to enhance osteoblast mineralization for scaffold-based bone regeneration Chunhui Jiang1, 2, Naagarajan Narayanan1, 2, Shihuan Kuang3, 4 and Meng Deng1, 2, 5, 6 1 Purdue University, Department of Agricultural and Biological Engineering, United States 2 Purdue University, Bindley Bioscience Center, United States 3 Purdue University, Department of Animal Sciences, United States 4 Purdue University, Center for Cancer Research, United States 5 Purdue University, School of Materials Engineering, United States 6 Purdue University, Weldon School of Biomedical Engineering, United States Introduction: Bone fracture has become prevalent in modern society, especially with an increasingly aging population. Current bone grafts procedures, including autografts and allografts, are hindered by multiple factors, such as limited supplies and inconsistent bone healing. Regenerative engineering, involving the integration of biomaterials with cell biology, emerges as a transdisciplinary strategy to induce bone regeneration. Poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds represent a promising biomimetic matrix system with the architectural and mechanical properties mimicking the natural bone environment. Recent advances in bone biology have suggested the important roles of Notch (Fig 1), a classical signaling pathway controlling cell fates and tissue development, on osteoblast differentiation[1]. The long term goal of this work is to develop osteoinductive biomaterials by regulating Notch signaling for enhanced osteogenic differentiation and mineralization. In the present work, we studied the effect of Notch inhibition on osteoblasts cultured on a biomimetic PLAGA scaffold by using a γ-secretase inhibitor (DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). It is hypothesized that PLAGA scaffolds combined with DAPT treatment will promote osteoblast mineralization. Materials and Methods: PLAGA 85:15 was purchased from Lakeshore Biomaterials. DAPT was purchased from R&D system. PLAGA microspheres (500–710 μm in diameter) were obtained using the emulsion-solvent evaporation method[2]. 3D scaffolds were fabricated by heat sintering at an optimized condition and characterized with SEM and micro-computed tomography. Compressive testing was conducted on the cylindrical scaffolds (10 mm in height and 5 mm in diameter, n=6) using an Instron machine. Primary rat osteoblasts were isolated and induced for osteogenic mineralization with PLAGA scaffolds[3]. In vitro studies were performed to investigate the osteoinductive potential of DAPT with the PLAGA scaffolds. Cell adhesion was evaluated using actin cytoskeleton staining kit (Millipore). Primary osteoblast mineralization was assessed by alizarin red staining. Results and Discussion: PLAGA microsphere scaffolds were fabricated at a sintering temperature of 90°C for 2h (Fig 2A). The scaffold showed a compressive modulus of ~214.MPa and a compressive strength of ~8.64 MPa, which were in the range of values for human trabecular bone. The interconnected porous structure was confirmed by SEM (Fig 2B) and micro-CT (Fig. 2C and 2D). These PLAGA scaffolds possessed 30% porosity with 100-350 μm pore size, which have been shown to encourage cell migration into the scaffold interior for homogeneous cell distribution. Primary osteoblasts were induced for osteogenic mineralization on PLAGA scaffolds with continuous DAPT administration. After 14 days induction, mature osteoblasts were characterized by actin cytoskeleton staining and alizarin red staining. Cytoskeletal actin staining of mature osteoblasts exhibited a spread cell morphology with DAPT treatment, similar to DMSO treatment (Fig 3A), suggesting that cells were well tolerated with PLAGA scaffolds combined with DAPT. Fig 3B showed that DAPT-treated PLAGA microsphere scaffolds promoted osteogenic mineralization. This is further confirmed by light microscope images, displaying a wide distribution pattern of mineralization with DAPT treatment compared to scarce mineralization of the adjoining area between microspheres in DMSO group (Fig 3C). Conclusion: We have successfully demonstrated the feasibility of employing Notch signalling pathway to enhance PLAGA scaffold-based bone regeneration. Our studies provide encouraging proof-of-principle evidences for harnessing Notch signaling pathway to facilitate biomaterial scaffold-based bone regeneration. Notch regulating scaffolds hold great promise for the development of osteoinductive bone graft substitutes. Showalter Trust; Purdue Research Foundation; Purdue Start-up Package

Modulation of Inflammatory Response and Induction of Bone Formation Based on Combinatorial Effects of Resveratrol.

The success of bone tissue engineering strategies critically depends on the rapid formation of a mature vascular network in the scaffolds after implantation. Conventional methods to accelerate the infiltration of host vasculature into the scaffolds need to consider the role of host response in regulation of bone tissue ingrowth and extent of vascularization. The long term goal of this study was to harness the potential of inflammatory response to enhance angiogenesis and bone formation in three dimensional (3D) scaffolds. Towards this goal, we explored the use of resveratrol, a natural compound commonly used in complementary medicine, to enable the concurrently (i) mediate M1 to M2 macrophage plasticity, (ii) impart natural release of angiogenic factors by macrophages and (iii) enhance osteogenic differentiation of human mesenchymal stem cells (hMSCs). We mapped the time-dependent response of macrophage gene expression as well as hMSC osteogenic differentiation to varying doses of resveratrol. The utility of this approach was evaluated in 3D poly (lactide-co-glycolide) (PLGA) sintered microsphere scaffolds for bone tissue engineering applications. Our results altogether delineate the potential to synergistically accelerate angiogenic factor release and upregulate osteogenic signaling pathways by "dialing" the appropriate degree of resveratrol release.

In vitro degradation and bioactivity of poly(propylene fumarate)/bioactive glass sintered microsphere scaffolds for bone tissue engineering

Abstract We developed degradable poly(propylene fumarate)/bioactive glass (PPF/BG) composite scaffolds based on a sintered microsphere technique and investigated the effects of BG content on the characteristics of these composite scaffolds. Immersion in a simulated body fluid (SBF) was used to evaluate the surface reactivity of composite scaffolds. The surface of composite scaffolds was covered with hydroxycarbonate apatite layer after 7 days of immersion. Ion concentration analyses revealed a decrease in P concentration and an increase in Si, Ca, and Sr concentrations in SBF immersed with composite scaffolds during the 3-week period. The Ca and P uptake rates decreased after 4 days of incubation. This coincided with the decrease of the Si release rate. These data lend support to the suggestion that the Si released from the BG content of scaffolds present in the polymer matrix was involved in the formation of the Ca-P layer. The evaluation of the in vitro degradation of composite microspheres revealed that the weight of scaffolds remained relatively constant during the first 3 weeks and then started to decrease slowly, losing 10.5% of their initial mass by week 12. Our results support the concept that these new bioactive, degradable composite scaffolds may be used for bone tissue engineering applications.

Evaluation of PLAGA/n-HA Composite Scaffold Bioactivity in vitro

Polymeric sintered microsphere scaffolds have shown their tremendous potential in bone tissue engineering applications due to their highly porous and interconnected three dimensional structure and excellent mechanical properties. While these scaffolds are able to support basic cellular activity after seeding cells on them, the bioactivity of these scaffolds in terms of enhancing the biological performance of stem cells during bone regeneration is still under satisfactory. We hypothesized that incorporation of bioactive addictive such as hydroxyapatite into these scaffolds could improve their bioactivity without sacrificing the bulk properties of the scaffolds. We have successfully incorporated nano-hydroxyapatite (n-HA) into poly (lactic acid-glycolic acid) (PLAGA) microsphere based scaffolds in our previous studies. Herein, we aimed to evaluate the bioactivity of PLAGA/n-HA composite scaffolds, with a focus on studying the mineralization of the scaffolds in vitro. The capability of inducing apatite formation in vitro was largely enhanced in the composite scaffolds compared to plain PLAGA scaffolds. More importantly, PLAGA/n-HA composite scaffolds have been shown to improve rabbit mesenchymal stem cells (RMSCs) proliferation, differentiation, and mineralization as compared to control PLAGA scaffolds. Taken together, introduction of n-HA appears to be an efficient approach to improve the bioactivity of PLAGA scaffolds for bone tissue engineering.

VEGF‐incorporated biomimetic poly(lactide‐co‐glycolide) sintered microsphere scaffolds for bone tissue engineering

Regenerative engineering approaches utilizing biomimetic synthetic scaffolds provide alternative strategies to repair and restore damaged bone. The efficacy of the scaffolds for functional bone regeneration critically depends on their ability to induce and support vascular infiltration. In the present study, three-dimensional (3D) biomimetic poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds were developed by sintering together PLAGA microspheres followed by nucleation of minerals in a simulated body fluid. Further, the angiogenic potential of vascular endothelial growth factor (VEGF)-incorporated mineralized PLAGA scaffolds were examined by monitoring the growth and phenotypic expression of endothelial cells on scaffolds. Scanning electron microscopy micrographs confirmed the growth of bone-like mineral layers on the surface of microspheres. The mineralized PLAGA scaffolds possessed interconnectivity and a compressive modulus of 402 ± 61 MPa and compressive strength of 14.6 ± 2.9 MPa. Mineralized scaffolds supported the attachment and growth and normal phenotypic expression of endothelial cells. Further, precipitation of apatite layer on PLAGA scaffolds resulted in an enhanced VEGF adsorption and prolonged release compared to nonmineralized PLAGA and, thus, a significant increase in endothelial cell proliferation. Together, these results demonstrated the potential of VEGF-incorporated biomimetic PLAGA sintered microsphere scaffolds for bone tissue engineering as they possess the combined effects of osteointegrativity and angiogenesis.

Novel mechanically competent polysaccharide scaffolds for bone tissue engineering

The success of the scaffold-based bone regeneration approach critically depends on the biomaterial's mechanical and biological properties. Cellulose and its derivatives are inherently associated with exceptional strength and biocompatibility due to their β-glycosidic linkage and extensive hydrogen bonding. This polymer class has a long medical history as a dialysis membrane, wound care system and pharmaceutical excipient. Recently cellulose-based scaffolds have been developed and evaluated for a variety of tissue engineering applications. In general porous polysaccharide scaffolds in spite of many merits lack the necessary mechanical competence needed for load-bearing applications. The present study reports the fabrication and characterization of three-dimensional (3D) porous sintered microsphere scaffolds based on cellulose derivatives using a solvent/non-solvent sintering approach for load-bearing applications. These 3D scaffolds exhibited a compressive modulus and strength in the mid-range of human trabecular bone and underwent degradation resulting in a weight loss of 10–15% after 24 weeks. A typical stress–strain curve for these scaffolds showed an initial elastic region and a less-stiff post-yield region similar to that of native bone. Human osteoblasts cultured on these scaffolds showed progressive growth with time and maintained expression of osteoblast phenotype markers. Further, the elevated expression of alkaline phosphatase and mineralization at early time points as compared to heat-sintered poly(lactic acid–glycolic acid) control scaffolds with identical pore properties affirmed the advantages of polysaccharides and their potential for scaffold-based bone regeneration.

Sintered Microsphere Scaffolds for Controlled Release and Tissue Engineering

Sintered Microsphere Scaffolds for Controlled Release and Tissue Engineering

Microsphere-based drug releasing scaffolds for inducing osteogenesis of human mesenchymal stem cells in vitro

In this study, in vitro osteogenesis was successfully achieved in human mesenchymal stem cells (hMSCs) by controlled release of the osteogenesis-inducing drugs dexamethasone, ascorbic acid (AA) and β-glycerophosphate (GP) from poly(lactic-co-glycolic acid) (PLGA) sintered microsphere scaffolds (SMS). We investigated the osteogenesis of human MSCs (hMSCs) on dexamethasone laden PLGA-SMS (PLGA-Dex-SMS), and dexamethasone, AA and GP laden PLGA-SMS (PLGA-Com-SMS). hMSCs cultured on the microsphere systems, which act as drug release vehicles and also promote cell growth/tissue formation—displayed a strong osteogenic commitment locally. The osteogenic commitment of hMSCs on the scaffolds were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real-time PCR and immunohistochemistry analysis. The results indicated hMSCs cultured on PLGA-Com-SMS exhibited superior osteogenic differentiation owing to significantly high phenotypic expression of typical osteogenic genes—osteocalcin (OC), type I collagen, alkaline phosphatase (ALP), and Runx-2/Cbfa-1, and protein secretion of bone-relevant markers such as osteoclast and type I collagen when compared with PLGA-Dex-SMS. In conclusion, by promoting osteogenic development of hMSCs in vitro, this newly designed controlled release system opens a new door to bone reparation and regeneration.

Functionalization of chitosan/poly(lactic acid‐glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering

Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 microg/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 microg/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications.

Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: A combined gene therapy–cell transplantation approach

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials.

Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide‐co‐glycolide) sintered microsphere scaffolds for bone tissue engineering

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.

Apatite nano-crystalline surface modification of poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering: implications for protein adsorption

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.

A preliminary report on a novel electrospray technique for nanoparticle based biomedical implants coating: Precision electrospraying

The compatibility and biological efficacy of biomedical implants can be enhanced by coating their surface with appropriate agents. For predictable functioning of implants in situ, it is often desirable to obtain an extremely uniform coating thickness without effects on component dimensions or functions. Conventional coating techniques require rigorous processing conditions and often have limited adhesion and composition properties. In the present study, the authors report a novel precision electrospraying technique that allows both degradable and nondegradable coatings to be placed. Thin metallic slabs, springs, and biodegradable sintered microsphere scaffolds were coated with poly(lactide-co-glycolide) (PLAGA) using this technique. The effects of process parameters such as coating material concentration and applied voltage were studied using PLAGA and poly(ethylene glycol) coatings. Morphologies of coated surfaces were qualitatively characterized by scanning electron microscopy. Qualitative observations suggested that the coatings were composed of particles of various size/shape and agglomerates with different porous architectures. PLAGA coatings of uniform thickness were observed on all surfaces. Spherical nanoparticle poly(ethylene glycol) coatings (462-930 nm) were observed at all concentrations studied. This study found that the precision electrospraying technique is elegant, rapid, and reproducible with precise control over coating thickness (mum to mm) and is a useful alternative method for surface modification of biomedical implants.

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.