High Molecular Weight Poly(glutamic acid) to Improve BMP2-Induced Osteogenic Differentiation.

FDA-approved bone morphogenetic protein 2 (BMP2) has serious side effects due to the super high dose requirement. Heparin is one of the most well-studied sulfated polymers to stabilize BMP2 and improve its functionality. However, the clinical use of heparin is questionable because of its undesired anticoagulant activity. Recent studies suggest that poly(glutamic acid) (pGlu) has the potential to improve BMP2 bioactivity with less safety concerns; however, the knowledge on pGlu's contribution remains largely unknown. Therefore, we aimed to study the role of pGlu in BMP2-induced osteogenesis and its potential application in bone tissue engineering. Our data, for the first time, indicated that both low (L-pGlu) and high molecular weight pGlu (H-pGlu) were able to significantly improve the BMP2-induced early osteoblastic differentiation marker (ALP) in MC3T3-E1 preosteoblasts. Importantly, the matrix mineralization was more rapidly enhanced by H-pGlu compared to L-pGlu. Additionally, our data indicated that only α-H-pGlu could significantly improve BMP2's activity, whereas γ-H-pGlu failed to do so. Moreover, both gene expression and mineralization data demonstrated that α-H-pGlu enabled a single dose of BMP2 to induce a high level of osteoblastic differentiation without multiple doses of BMP2. To study the potential application of pGlu in tissue engineering, we incorporated the H-pGlu+BMP2 nanocomplexes into the collagen hydrogel with significantly elevated osteoblastic differentiation. Furthermore, H-pGlu-coated 3D porous gelatin and chitosan scaffolds significantly enhanced osteogenic differentiation through enabling sustained release of BMP2. Thus, our findings suggest that H-pGlu is a promising new alternative with great potential for bone tissue engineering applications.

MicroRNA-200c Release from Gelatin-Coated 3D-Printed PCL Scaffolds Enhances Bone Regeneration.

The fabrication of clinically relevant synthetic bone grafts relies on combining multiple biodegradable biomaterials to create a structure that supports the regeneration of defects while delivering osteogenic biomolecules that enhance regeneration. MicroRNA-200c (miR-200c) functions as a potent osteoinductive biomolecule to enhance osteogenic differentiation and bone formation; however, synthetic tissue-engineered bone grafts that sustain the delivery of miR-200c for bone regeneration have not yet been evaluated. In this study, we created novel, multimaterial, synthetic bone grafts from gelatin-coated 3D-printed polycaprolactone (PCL) scaffolds. We attempted to optimize the release of pDNA encoding miR-200c by varying gelatin types, concentrations, and polymer crosslinking materials to improve its functions for bone regeneration. We revealed that by modulating gelatin type, coating material concentration, and polymer crosslinking, we effectively altered the release rates of pDNA encoding miR-200c, which promoted osteogenic differentiation in vitro and bone regeneration in a critical-sized calvarial bone defect animal model. We also demonstrated that crosslinking the gelatin coatings on the PCL scaffolds with low-concentration glutaraldehyde was biocompatible and increased cell attachment. These results strongly indicate the potential use of gelatin-based systems for pDNA encoding microRNA delivery in gene therapy and further demonstrate the effectiveness of miR-200c for enhancing bone regeneration from synthetic bone grafts.

Vanillin-bioglass cross-linked 3D porous chitosan scaffolds with strong osteopromotive and antibacterial abilities for bone tissue engineering.

Chitosan scaffolds crosslinked by current methods insufficiently meet the demands of bone tissue engineering applications. We developed a novel effective crosslinking technique by using the natural and safe vanillin together with bioglass microparticles to generate an antibacterial, osteoconductive, and mechanically robust 3D porous chitosan-vanillin-bioglass (CVB) scaffold. In addition to the significantly improved mechanical properties, the CVB scaffolds had high porosity (>90%) and interconnected macroporous structures. Our data suggested that the crosslinking mainly resulted from the Schiff base reactions between the aldehydes of vanillin and amines of chitosan, together with the hydrogen and ionic bonds formed within them. Importantly, the CVB scaffolds not only showed good biocompatibility, bioactivity, and strong antibacterial ability but also significantly promoted osteoblastic differentiation, mineralization in vitro, and ectopic bone formation in vivo. Thus, the CVB scaffolds hold great promise for bone tissue engineering applications based on their robust mechanical properties, osteoconductivity, and antibacterial abilities.

Biomimetic Nanofibrous 3D Materials for Craniofacial Bone Tissue Engineering.

Repair of large bone defects using biomaterials-based strategies has been a significant challenge due to the complex characteristics required for tissue regeneration, especially in the craniofacial region. Tissue engineering strategies aimed at restoration of function face challenges in material selection, synthesis technique, and choice of bioactive factor release in combination with all aforementioned facets. Biomimetic nanofibrous (NF) scaffolds are attractive vehicles for tissue engineering due to their ability to promote endogenous bone regeneration by mimicking the shape and chemistry of natural bone extracellular matrix (ECM). To date, several techniques for generation of biomimetic NF scaffolds have been discovered, each possessing several advantages and drawbacks. This spotlight highlights two of the more popular techniques for biomimetic NF scaffold synthesis: electrospinning and thermally-induced phase separation (TIPS), covering development from inception in each technique as well as discussing the most recent innovations in each fabrication method.

Nanoclay Promotes Mouse Cranial Bone Regeneration Mainly through Modulating Drug Binding and Sustained Release.

Nanoclay (Nanosilicates, NS) is appearing as an intriguing 2D nanomaterial for bone tissue engineering with multiple proposed functions, e.g., intrinsic osteoinductivity, improving mechanical properties, and drug release capacity. However, the mechanism of NS for in vivo bone regeneration has been hardly defined so far. This knowledge gap will significantly affect the design/application of NS-based biomaterials. To determine the role of NS in osteoblastic differentiation and bone formation, we used the mouse calvarial-derived pre-osteoblasts (MC3T3-E1) and a clinically-relevant mouse cranial bone defect model. Instead of a hydrogel, we prepared biomimetic 3D gelatin nanofibrous scaffolds (GF) and NS-blended composite scaffolds (GF/NS) to determine the essential role of NS in critical low-dose (0.5 µg per scaffold) of BMP2-induced cranial bone regeneration. In contrast to "osteoinductivity", our data indicated that NS could enable single-dose of BMP2, promoting significant osteoblastic differentiation while multiple-dose of BMP2 (without NS) was required to achieve similar efficacy. Moreover, our release study revealed that direct binding to NS in GF scaffolds provided stronger protection to BMP2 and sustained release compared to GF/NS composite scaffolds. Consistently, our in vivo data indicated that only BMP2/NS direct binding treatment was able to repair the large mouse cranial bone defects after 6 weeks of transplantation while neither BMP2, NS alone, nor BMP2 released from GF/NS scaffolds was sufficient to induce significant cranial bone defect repair. Therefore, we concluded that direct nanoclay-drug binding enabled sustained release is the most critical contribution to the significantly improved bone regeneration compared to other possible mechanisms based on our study.

One-pot porogen free method fabricated porous microsphere-aggregated 3D PCL scaffolds for bone tissue engineering.

Three-dimensional (3D) scaffolds with interconnected, hierarchically structured pores, and biomimetic nanostructures are desirable for tissue engineering, where preparation with a facile and biocompatible strategy remains challenging. In the present work, an innovative porous microspheres-aggregated 3D PCL scaffold with macropores, micropores, and nanofibrous-like structures was fabricated through a one-pot thermally induced phase separation (TIPS) method without the use of any porogen or specific instruments. Importantly, the porosity, pore size, and mechanical properties of our scaffolds were tailorable through tuning of the polymer concentration. Interestingly, the bioactivity of our 3D PCL scaffolds was significantly improved, as abundant apatite-like layers were formed on the 3D porous scaffolds, while no obvious apatite was observed on the 2D flat PCL film. Moreover, the high surface area attributed to the hierarchical macro/micro/nanostructure enabled our 3D porous scaffold to serve as a drug delivery depot for sustained release of both small molecule drug (phenamil) and protein (BMP2). In addition to sustained drug release, the hierarchical structure and high mechanical properties also contribute to significantly improving BMP2-induced osteogenic differentiation. In summary, we developed a novel PCL porous scaffold through a facile, one-pot TIPS method and demonstrated its promising potential application in large bone defect repair.

Tailoring weight ratio of PCL/PLA in electrospun three-dimensional nanofibrous scaffolds and the effect on osteogenic differentiation of stem cells.

Three-dimensional (3D) scaffolds as artificial ECMs have been extensively studied to mimic the critical features of natural ECMs. To develop more clinically relevant 3D scaffolds, electrospun nanofibrous scaffolds with different weight ratios of PCL/PLA (i.e., 100/0, 60/40, and 20/80) were fabricated via the thermally induced (nanofiber) self-agglomeration (TISA) method. The hypothesis was that, with the weight ratio increase of stiffer and more bioactive PLA in the 3D PCL/PLA blend scaffolds, the osteogenic differentiation of human mesenchymal stem cells (hMSCs) would be enhanced. The results indicated that, all of the 3D scaffolds were elastic/resilient and possessed interconnected and hierarchical pores with sizes from sub-microns to ∼300 µm; therefore, the morphological structures of these scaffolds were similar to those of natural ECMs. The PLA80 scaffolds exhibited the best overall properties in terms of density, porosity, water absorption capacity, mechanical properties, bioactivity, and cell viability. Furthermore, with increasing the PLA weight ratio, the alkaline phosphatase (ALP) activity, calcium content, and gene expression level were also increased, probably due to the improved stiffness/bioactivity of scaffold. Hence, the novel 3D electrospun PLA80 nanofibrous scaffold might be desired/favorable for the osteogenic differentiation of hMSCs.

Functionalization of PCL-3D Electrospun Nanofibrous Scaffolds for Improved BMP2-Induced Bone Formation.

Bone morphogenic protein 2 (BMP2) is a key growth factor for bone regeneration, possessing FDA approval for orthopedic applications. BMP2 is often required in supratherapeutic doses clinically, yielding adverse side effects and substantial treatment costs. Considering the crucial role of materials for BMPs delivery and cell osteogenic differentiation, we devote to engineering an innovative bone-matrix mimicking niche to improve low dose of BMP2-induced bone formation. Our previous work describes a novel technique, named thermally induced nanofiber self-agglomeration (TISA), for generating 3D electrospun nanofibrous (NF) polycaprolactone (PCL) scaffolds. TISA process could readily blend PCL with PLA, leading to increased osteogenic capabilities in vitro, however, these bio-inert synthetic polymers produced limited BMP2-induced bone formation in vivo. We therefore hypothesize that functionalization of NF 3D PCL scaffolds with bone-like hydroxyapatite (HA) and BMP2 signaling activator phenamil will provide a favorable osteogenic niche for bone formation at low doses of BMP2. Compared to PCL-3D scaffolds, PCL/HA-3D scaffolds demonstrated synergistically enhanced osteogenic differentiation capabilities of C2C12 cells with phenamil. Importantly, in vivo studies showed this synergism was able to generate significantly increased new bone in an ectopic mouse model, suggesting PCL/HA-3D scaffolds act as a favorable synthetic extracellular matrix for bone regeneration.

Three dimensional electrospun PCL/PLA blend nanofibrous scaffolds with significantly improved stem cells osteogenic differentiation and cranial bone formation.

Nanofibrous scaffolds that are morphologically/structurally similar to natural ECM are highly interested for tissue engineering; however, the electrospinning technique has the difficulty in directly producing clinically relevant 3D nanofibrous scaffolds with desired structural properties. To address this challenge, we have developed an innovative technique of thermally induced nanofiber self-agglomeration (TISA) recently. The aim of this work was to prepare (via the TISA technique) and evaluate 3D electrospun PCL/PLA blend (mass ratio: 4/1) nanofibrous scaffolds having high porosity of ∼95.8% as well as interconnected and hierarchically structured pores with sizes from sub-micrometers to ∼300 µm for bone tissue engineering. The hypothesis was that the incorporation of PLA (with higher mechanical stiffness/modulus and bioactivity) into PCL nanofibers would significantly improve human mesenchymal stem cells (hMSCs) osteogenic differentiation in vitro and bone formation in vivo. Compared to neat PCL-3D scaffolds, PCL/PLA-3D blend scaffolds had higher mechanical properties and in vitro bioactivity; as a result, they not only enhanced the cell viability of hMSCs but also promoted the osteogenic differentiation. Furthermore, our in vivo studies revealed that PCL/PLA-3D scaffolds considerably facilitated new bone formation in a critical-sized cranial bone defect mouse model. In summary, both in vitro and in vivo results indicated that novel 3D electrospun PCL/PLA blend nanofibrous scaffolds would be strongly favorable/desired for hMSCs osteogenic differentiation and cranial bone formation.

Electrospun polycaprolactone 3D nanofibrous scaffold with interconnected and hierarchically structured pores for bone tissue engineering.

For the first time, electrospun polycaprolactone (PCL) 3D nanofibrous scaffold has been developed by an innovative and convenient approach (i.e., thermally induced nanofiber self-agglomeration followed by freeze drying), and the scaffold possesses interconnected and hierarchically structured pores including macropores with sizes up to ≈300 µm. The novel PCL 3D scaffold is soft and elastic with very high porosity of ≈96.4%, thus it is morphologically/structurally similar to natural extracellular matrix and well suited for cell functions and tissue formation. The in vitro studies reveal that the scaffold can lead to high cell viability; more importantly, it is able to promote more potent BMP2-induced chondrogenic than osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Consistent to the in vitro findings, the in vivo results indicate that the electrospun PCL 3D scaffold acts as a favorable synthetic extracellular matrix for functional bone regeneration through the physiological endochondral ossification process.