Microfluidic fabrication of microcarriers with sequential delivery of VEGF and BMP-2 for bone regeneration

Abstract

Wound instability and poor functional vascularization in bone tissue engineering lead to lack of tissue integration and ultimate failure of engineered grafts. In order to harness the regenerative potential of growth factors and stimulate bone healing, present study aims to design multifunctional cell therapy microcarriers with the capability of sequential delivery of essential growth factors, bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF). An on-chip double emulsion method was implemented to generate monodisperse VEGF encapsulated microcarriers. Bio-inspired poly(3,4-dihydroxyphenethylamine) (PDA) was then functionalized to the microcarriers surface for BMP-2 conjugation. The microcarriers were seeded with mesenchymal stem cells (MSCs) using a dynamic culture technique for cells expansion. Finally, the microcarriers were incorporated into an injectable alginate-RGD hydrogel laden with endothelial cells (ECs) for further analysis. The DNA and calcium content, as well as ALP activity of the construct were analyzed. The confocal fluorescent microscopy was employed to monitor the MSCs and tunneling structure of ECs. Eventually, the capability of developed microcarriers for bone tissue formation was examined in vivo. Microfluidic platform generated monodisperse VEGF-loaded PLGA microcarriers with size-dependent release patterns. Microcarriers generated with the on-chip technique showed more sustained VEGF release profiles compared to the conventional bulk mixing method. The PDA functionalization of microcarriers surface not only provided immobilization of BMP-2 with prolonged bioavailability, but also enhanced the attachment and proliferation of MSCs. Dynamic culturing of microcarriers showcased their great potential to boost MSCs population required for stem cell therapy of bone defects. ALP activity and calcium content analysis of MSCs-laden microcarriers loaded into injectable hydrogels revealed their capability of tunneling formation, vascular cell growth and osteogenic differentiation. The in vivo histology and real-time polymerase chain reaction analysis revealed that transplantation of MSCladen microcarriers supports ectopic bone formation in the rat model. The presented approach to design bioactive microcarriers offer sustained sequential delivery of bone ECM chemical cues and offer an ideal stabilized 3D microenvironment for patient-specific cell therapy applications. The proposed methodology is readily expandable to integrate other cells and cytokines in a tuned spatiotemporal manner for personalized regenerative medicine. Bone healing is a multifaceted developmental process which involves multiple signaling molecules in a specific localization and orchestration to induce vascularization and osteogenesis 1-3. Vascularization plays a leading role in tissue repair and regeneration 4,5. However, conventional constructs for bone tissue engineering are poorly vascularized, which typically leads to their weak integration and survival 6. On the other hand, slow penetration of the...

Microfluidic Synthesis of Indomethacin-Loaded PLGA Microparticles Optimized by Machine Learning

“…Microfluidics has offered an effective alternative to traditional methods to fabricate polymeric microparticles. Nowadays, microfluidics is an effective tool to generate microparticles with high monodispersity, precisely tunable structures, and excellent encapsulation efficiency ( Damiati et al, 2018 ; Dashtimoghadam et al, 2020 ). Microfluidic chips are either lab-made or commercial products made of glass, polymers, or polydimethylsiloxane (PDMS), and consist of rectangular microchannels in different dimensions that are constructed by lithography ( Damiati et al, 2018 ; Dewandre et al, 2020 ).…”

Iron–Quercetin Complex Preconditioning of Human Peripheral Blood Mononuclear Cells Accelerates Angiogenic and Fibroblast Migration: Implications for Wound Healing

“…There is growing evidence that angiogenesis is a critical process in tissue repair and regeneration. Dashtimoghadam et al demonstrated that multifunctional cell therapy microcarriers containing mesenchymal stem cells (MSCs), endothelial cells, and vascular endothelial growth factor (VEGF) enhance the regeneration of bone tissue in vivo [43]. New blood vessel formation is a prominent process during tissue repair.…”

A Dual Peptide Sustained-Release System Based on Nanohydroxyapatite/Polyamide 66 Scaffold for Synergistic-Enhancing Diabetic Rats’ Fracture Healing in Osteogenesis and Angiogenesis

“…Some studies have reported that bone regeneration can be significantly improved by the combined BMP-2 and VEGF growth factors. Dashtimoghadam et al (2020) demonstrated conjugation of BMP-2 growth factor onto monodisperse polymeric microcarriers encapsulating VEGF growth factor, which displayed an additive effect on bone regeneration. Moreover, in the study of Godoy-Gallardo et al (2020) , BMP-2 was bound onto the inner polydopamine layer, whereas VEGF was immobilized onto the outer one, and the two growth factors played a synergistic effect on bone regeneration.…”