The major challenge in reconstruction of large bone defects is that the maturation of osteogenic and vasculogenic cells require complementary microenvironments.
The aim of this project is to develop a 3D multilayer cell-laden composite construct with spatially organized microchannels to mimic the "osteoblastic- vascular" niche in the bone marrow, and to address the issues of tissue architecture and cell microenvironment. In bone tissue, the soft permissive vasculoinductive microenvironment of the marrow stroma supports vasculogenesis while the osteoinductive endosteal layer bound to the osseous tissue supports mineralization and bone formation. The hypotheses are: a) an osteoinductive high modulus hydrogel with and long-degradation time (SPELA gel) provides a microenvironment for mineralization of marrow stromal cells (MSCs);b) a high compliance hydrogel with short degradation (GelMA gel) provides a permissive microenvironment for vascularization of endothelial progenitor cells (EPCs);c) microchannels of the soft GelMA gel patterned in the SPELA gel provide a permissive and instructive "osteoblastic-vascular" niche for concurrent vascularization and mineralization in the composite matrix;and d) the micropatterning process can be repeated to produce a 3D multilayer construct. We propose the following aims to engineer and evaluate the cellular constructs for regeneration of bone segments.
In Aim 1. 1, we will synthesize the SPELA hydrogel with short lactide segments as a degradable matrix with robust compressive modulus to support encapsulation and mineralization of MSCs.
In Aim 1. 2, we will supplement the SPELA hydrogel with osteoinductive rhBMP-2 protein grafted to self-assembled nanoparticles to prevent migration of the protein and confine its osteoinductivity to the SPELA matrix.
In Aim 1. 3, we will synthesize a gelatin-based GelMA hydrogel as a permissive matrix to support vasculogenic differentiation and maturation of bone marrow derived EPCs and MSCs and vessel formation.
In Aim 2. 1, we will fabricate microchannels of EPC/MSC- seeded GelMA gel in MSC-seeded SPELA hydrogel to form a "gel-in-gel" tissue layer with spatially organized microvessels in the mineralizing SPELA hydrogel.
In Aim 2. 2, we will engineer macroporous tissue layers, integrate the layers into 3D multilayer constructs with spatially organized microchannels, and determine viability of the embedded cells in the central part of the construct.
In Aim 2. 3, we will evaluate the 3D multilayer cell-laden constructs with respect to mineralization and vascularization in vitro.
In Aim 3, the patterned cell-laden 3D constructs will be evaluated in vivo in rat segmental femur defect for the extent of bone formation and healing. This is a clinically viable approach as MSCs and EPCs can be isolated from the bone marrow of the patient and embedded in the construct prior to implantation in a large bone defect.
Approximately 6.2 million fractures in the US annually require intervention in the form of bone graft to aid skeletal repair and achieve union. This project aims to develop a 3D multilayer cellular construct with spatially organized microchannels to address the issue of vascularization in the interior parts of the implant. The patterned multi-cellular approach adapted is clinically viable as the bone defect is implanted with a construct that is embedded with cells isolated from the patient's bone marrow.
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