A Modular Approach To The Engineering Of A Centimeter-Sized Bone Tissue Construct By Microfabrication

Tissue engineering holds great promise in generating functional replacements for tissue repair/regeneration. However, in traditional processes by seeding cells onto pre-fabricated scaffolds, there exists a mass transfer limit, which results in preferential cell proliferation and extracellular matrix (ECM) deposition on the outer region of scaffolds, and nutrition deprivation and severe cell death at the core. As a result, the dimension of engineered tissue constructs has been largely restricted within millimeters, which is obviously not able to meet the clinical demands, such as in a large bone fracture. To address this issue, we had proposed a microtissue assembly strategy (a modular approach) to fabricate large tissue constructs through the assembly of microtissues, which essentially recapitulates the basic functional unit of native tissues/organs. In this study, with objective to engineering large bone tissue structures, we explored to develop a methodology to fabricate bone-like microtissues with human amniotic mesenchymal stem cells (hAMSCs) and microcarriers, and then to establish a process to assemble these microtissues into viable centimeter-sized bone tissue constructs..First, a dynamic suspension culture system of human amniotic hAMSCs on microcarriers was set up in spinner flasks. A series of process parameters including microcarrier type and concentration, cell density, and stirring speed were examined concerning growth and metabolism of hAMSCs. It was found that CultiSpher S supported cell proliferation better than Cytopore 2. When hAMSCs were seeded at 5×104 cells/ml onto CultiSpher S microcarriers (2 mg/ml) in growth medium with a stirring speed of 50 rpm. hAMSCs quickly entered the exponential growth phase and reached a stationary phase on day 8, with a maximum density of (29.6±3.7)×106 cells/cm3, which represented 14.7±1.8 folds of expansion.Then, a two-stage culture strategy was devised to generate bone-like microtissues by allowing the expansion of hAMSCs on microcarriers at the first stage and then inducing osteogenic differentiation simply by medium switch at the second stage in the same spinner flask. The results showed that hAMSCs quickly entered the exponential growth phase post seeding and expanded for about 15.0 folds after 8 d with a maximum density of (29.3±2.2)×106 cells/cm3. After that, growth medium was replaced with osteogenic medium and cultured for additional 20 d. A significant increase in both ALP activity and calcium content was observed in these microtissues. Hence, with the two-stage culture, hAMSCs could proliferate on CultiSpher S microcarriers and be induced towards osteogenic differentiation, generating bone-like modular tissues for subsequent fabrication of macroscopic bone constructs.It was subsequently demonstrated that after a 7-d perfusion culture in a cylindrical perfusion culture chamber in osteogenic medium, these microtissues readily assembled into a centimeter-sized construct (diameterxheight:2.0 cm×1.0 cm). Both close stacking of microtissues and abundant bone-characteristic ECM were observed on the outer surface of the constructs; inside the constructs, viable cells were evenly distributed, microcarriers were loosely attached and there were some void space left without cells and ECM; both ALP activity and mineralization (Alizarin red S) were detected throughout the cross-section of the constructs. These results suggested the feasibility of engineering a centimeter-sized bone tissue construct by following the microtissue assembly process.In order to further increase cell density and ECM deposition in engineered macroscopic tissue construct, thus improving both integrity and osteogenic characteristics, effects of small molecules including sodium ascorbate and mercaptoethanol on the growth and differentiation of hAMSCs were first investigated in monolayer culture in culture plates. It was shown that sodium ascorbate inhibited growth of hAMSCs in a dose-dependent manner and mercaptoethanol was able to stimulate cell growth at a concentration between 50～100 μmol/L. In addition, sodium ascorbate at a concentration ≥100 μg/ml inhibited the osteogenic differentiation of hAMSCs, while supplementation of mercaptoethanol (≥50 μg/ml) could promote the mineral deposition.Accordingly, in both microtissue fabrication and assembly, sodium ascorbate and mercaptoethanol both at 50μg/ml were supplemented in the media and cell proliferation, ECM deposition, osteogenic differeniation as well as properties of macroscopic tissue constructs were evaluated. It turned out that for microtissues, sodium ascorbate did not exert any significant influence while mercaptoethanol not only stimulated cell proliferation, but also promoted the aggregation of cell-laden microcarriers into a size of 800-900 μm, showing significant expression of bone related markers. After 7 d of perfuson assembly, cylindrical constructs with a dimension of 2.0 cm×1.0 cm (diameterxheight) was obtained, showing higher cell density, viability and ECM production in mercaptoethanol-supplemented condition than others. These results proved that supplementation of a small molecule mercaptoethanol could improve the efficiency of the microtissue assembly process.Finally, effects of in vitro perfusion culture and subcutaneous implantation on tissue properties were examined. It was found that when perfusion culture time was extended from 1 w to 2 w, further improvments in cell viability, microstructure and osteogenic differentiation of hAMSCs were achieved. Importantly, the macroscopic tissue constructs well maintained gross morphology, cell viability and bone-like ECM production with significant infiltration of blood vessels after subcutaneous implantation of in nude mice for both 6 w and 12 w.Taken together, in the present study, by using hAMSCs and CuitiSpher S microcarriers, a two-stage culture integrating cell growth and differentiation in a spinner flask was developed for fabricating bone-like microtissues. In addition, it was found that cell density and ECM deposition in microtissues could be promoted by supplementation of a small molecule mercaptoethanol. By using a perfusion culture system, the assembly of these microtissues could be successfully achieved, generating integrated tissue constructs with a centimeter size, high cell viability and uniform spatial distribution of mineralized bone-like matrix, which was shown to maintain tissue characteristics in the ectopic site in vivo. This study demonstrates that the microtissue assembly process is promising for tissue engineering and provides a novel strategy for engineering clinical-relevant large tissue replacements.