Lorenz Meinel

Growth factor gradients via microsphere delivery in biopolymer scaffolds for osteochondral tissue engineering

Temporally and spatially controlled delivery of growth factors in polymeric scaffolds is crucial for engineering composite tissue structures, such as osteochondral constructs. In the present study, microsphere-mediated growth factor delivery in polymer scaffolds and its impact on osteochondral differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Two growth factors, bone morphogenetic protein 2 (rhBMP-2) and insulin-like growth factor I (rhIGF-I), were incorporated as a single concentration gradient or reverse gradient combining two factors in the scaffolds. To assess the gradient making system and the delivery efficiency of polylactic-co-glycolic acid (PLGA) and silk fibroin microspheres, initially an alginate gel was fabricated into a cylinder shape with microspheres incorporated as gradients. Compared to PLGA microspheres, silk microspheres were more efficient in delivering rhBMP-2, probably due to sustained release of the growth factor, while less efficient in delivering rhIGF-I, likely due to loading efficiency. The growth factor gradients formed were shallow, inducing non-gradient trends in hMSC osteochondral differentiation. Aqueous-derived silk porous scaffolds were used to incorporate silk microspheres using the same gradient process. Both growth factors formed deep and linear concentration gradients in the scaffold, as shown by enzyme-linked immunosorbent assay (ELISA). After seeding with hMSCs and culturing for 5 weeks in a medium containing osteogenic and chondrogenic components, hMSCs exhibited osteogenic and chondrogenic differentiation along the concentration gradients of rhBMP-2 in the single gradient of rhBMP-2 and reverse gradient of rhBMP-2/rhIGF-I, but not the rhIGF-I gradient system, confirming that silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. This novel silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs.

Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

Non-invasive time-lapsed monitoring and quantification of engineered bone-like tissue

The formation of bone-like tissue from human mesenchymal stem cells (hMSC) cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified over 44xa0days in culture using non-invasive time-lapsed micro-computed tomography (μCT). Each construct was imaged nine times inxa0situ. From μCT imaging, detailed morphometrical data on bone volume density, surface-to-volume ratio, trabecular thickness, trabecular spacing, and the structure model index and tissue mineral density were obtained. μCT irradiation did not impact the osteogenic performance of hMSCs based on DNA content, alkaline phosphatase activity, and calcium deposition when compared to non-exposed control samples. Bone-like tissue formation initiated at day 10 of the culture with the deposition of small mineralized clusters. Tissue mineral density increased linearly over time. The surface-to-volume ratio of the bone-like tissues converged asymptotically to 26xa0mm−1. Although in vitro formation of bone-like tissue started from clusters, the overall bone volume was not predictable from the time, number, and size of initially formed bone-like clusters. Based on microstructural analysis, the morphometry of the tissue-engineered constructs was found to be in the range of human trabecular bone. In future studies, non-invasive, time-lapsed monitoring may enable researchers to culture tissues in vitro, right until the development of a desired morphology is accomplished. Our data demonstrate the feasibility of qualitatively and quantitatively detailing the spatial and temporal mineralization of bone-like tissue formation in tissue engineering.

Biocompatibility and osteoconduction of macroporous silk fibroin implants in cortical defects in sheep

The goal of the presented study was to compare the biocompatibility and cellular responses to porous silk fibroin (SF) scaffolds produced in a water-based (UPW) or a solvent based process (HFIP) using two different SF sources. For that reason, four different SF scaffolds were implanted (n=6) into drill hole defects in the cancellous bone of the sheep tibia and humerus. The scaffolds were evaluated histologically for biocompatibility, cell-material interaction, and cellular ingrowth. New bone formation was observed macroscopically and histologically at 8 weeks after implantation. For semiquantitative evaluation, the investigated parameters were scored and statistically analyzed (factorial ANOVA). All implants showed good biocompatibility as evident by low infiltration of inflammatory cells and the absent encapsulation of the scaffolds in connective tissue. Multinuclear foreign body giant cells (MFGCs) and macrophages were present in all parts of the scaffold at the material surface and actively degrading the SF material. Cell ingrowth and vascularization were uniform across the scaffold. However, in HFIP scaffolds, local regions of void pores were present throughout the scaffold, probably due to the low pore interconnectivity in this scaffold type in contrast to UPW scaffolds. The amount of newly formed bone was very low in both scaffold types but was more abundant in the periphery than in the center of the scaffolds and for HFIP scaffolds mainly restricted to single pores.

Design and validation of a novel bioreactor principle to combine online micro-computed tomography monitoring and mechanical loading in bone tissue engineering

Mechanical loading plays an important role in bone remodeling in vivo and, therefore, has been suggested as a key parameter in stem cell-based engineering of bone-like tissue in vitro. However, the optimization of loading protocols during stem cell differentiation and subsequent bone-like tissue formation is challenged by multiple input factors, which are difficult to control and validate. These include the variable cellular performance of cells harvested from different patients, nonstandardized culture media components, the choice of the biomaterial forming the scaffold, and its morphology, impacting a broader validity of mechanical stimulation regimens. To standardize the cell culture of bone-like tissue constructs, we suggest the involvement of time-lapsed feedback loops. For this purpose we present a prototype bioreactor that combines online, nondestructive monitoring using micro-computed tomography and direct mechanical loading of three-dimensional tissue engineering constructs. Validation of this system showed displacement steps down to 1 microm and cyclic sinusoidal loadings of up to 10 Hz. Load detection resolution was 0.01 N, and micro-computed tomography data were of high quality. For the first time, the developed bioreactor links time-lapsed, nondestructive, and dynamic imaging with mechanical stimulation, designed for cell culture under sterile conditions. This system is believed to substantially improve todays experimental options to study and optimize osteogenic stem cell culture and differentiation at the interface with mechanical stimulation.