Centimeter-Sized Biomimetic Bone Constructs Fabricated Via BMP-Collagen Microcarriers And BMSC-Gelatin Microspheres

Bottom-up approaches has been widely used to fabricate large-sized tissue for regeneration medicine, especial to bone tissue engineering. Herein, we construct centimeter-sized bone-like tissue with rat bone mesenchymal stem cell(BMSC)-gelatin microspheres(BGM) as cell-culture modules and bone morphogenetic protein(BMP)-collagen microcarriers(BCM) as differentiation-control modules.BGM with a high cell density were prepared in the first 7 days in the spinner flask. Microcarriers(2 mg/m L) were seeded with BMSCs at a density of 5X104/ml cells/ m L in growth medium in a 250-m L siliconized spinner flask(Corning, USA) using an intermittent stirring regime(3 min agitation at 30 rpm followed by 30 min static, which was repeated for 8 h). It was demonstrated that BMSC were efficiently seeded onto Culti Spher S microcarriers(made of porcine gelatin) in a spinner flask and quickly proliferated while retaining a great viability.BCM were prepared by collagen binding domain(CBD)-BMP binding with collagen microhydrgel. Linear ordered collagen has proper biocompatibility and there was also no mutagenicity of the LOCS in vitro. CBD-BMP, fused a collagen-binding domain(CBD) to bone morphogenetic protein(BMP), specifically bind to collagen binding domain(CBD) and CBD does not affect the activity of the BMP.In in vitro fabrication, BGM and BCM were mixed into the perfusion culture system to form bone-like construct in 10 days. After a 10-day perfusion culture, these modular tissues readily assembled into a centimeter-sized construct(diameter×height: 2 cm×1.1cm). Both good cell viability and fairly homogenous distribution of cellular content and bone-characteristic ECM within the macrotissue were elaborated. Compared with the sample without BCM, increased mineral deposition and ALP activity(Alizarin red stain 、ALP stain、quantification of calcium content) was found in the BCM sample.In vivo fabrication, BGM and BCM were injected into rat back to construct bone-like tissue in 30 days. At 30 days animals were sacrificed with injecting overdose sodium pentobarbital and the implants were excised. Homogeneous bone formation was observed after 30 days in the test group. HE stain, ALP stain,Alizarin red stain,quantification of calcium content and micro-computed tomography(micro-CT) confirm that BCM could direct BGM to grow into bone-like tissue.Our results suggest that these strategies, assembling cell-culture modules and function-control modules into aimed tissue in the perfusion culture system, could be used to fabricate a macroscopic tissue for tissue engineering. Biomimetic Bone Constructs Fabricated via BMP-collagen Microcarriers and BMSC-gelatin Microspheres can be used as bone graft. It will be welcomed by patients that needing bone grafting in the future.