Biomaterials derived from silk fibrion prepared by aqueous (AB) and organic (HFIP) solvent based processes, along with collagen (COL) and poly-lactic acid (PLA) based scaffolds were studied in vitro and in vivo for their utility in adipose tissue engineering strategies. For in vitro studies, human bone marrow and adipose-derived mesenchymal stem cells (hMSCs and hASCs) were seeded on the various biomaterials and cultured for 21 days in the presence of adipogenic stimulants (AD) or maintained as noninduced controls. Alamar Blue analysis revealed each biomaterial supported initial attachment of hMSCs and hASCs to similar levels for all matrices except COL in which higher levels were observed. hASCs and hMSCs cultured on all biomaterials in the presence of AD showed significant upregulation of adipogenic mRNA transcript levels (LPL, GLUT4, FABP4, PPARγ, adipsin, ACS) to similar extents when compared to noninduced controls. Similarly Oil-Red O analysis of hASC or hMSC-seeded scaffolds displayed substantial amounts of lipid accumulating adipocytes following cultivation with AD. The data revealed AB and HFIP scaffolds supported similar extents of lipid accumulating cells while PLA and COL scaffolds qualitatively displayed lower and higher extents by comparison, respectively. Following a 4 week implantation period in a rat muscle pouch defect model, both AB and HFIP scaffolds supported in vivo adipogenesis either alone or seeded with hASCs or hMSCs as assessed by Oil-Red O analysis, however the presence of exogenous cell sources substantially increased the extent and frequency of adipogenesis observed. In contrast, COL and PLA scaffolds underwent rapid scaffold degradation and were irretrievable following the implantation period. The results suggest that macroporous 3D AB and HFIP silk fibroin scaffolds offer an important platform for cell-based adipose tissue engineering applications, and in particular, provide longer-term structural integrity to promote the maintenance of soft tissue in vivo.

Bone is a dynamic tissue that is able to sense and adapt to mechanical stimuli by modulating its mass, geometry, and structure. Bone marrow stromal cells (BMSCs) are known to play an integral part in bone formation by providing an osteoprogenitor cell source capable of differentiating into mature osteoblasts in response to mechanical stresses. Characteristics of the in vivo bone environment including the three dimensional (3-D) lacunocanalicular structure and extracellular matrix composition have previously been shown to play major roles in influencing mechanotransduction processes within bone cells. To more accurately model this phenomenon in vitro, we cultured human BMSCs on 3-D, partially demineralized bone scaffolds in the presence of four-point bending loads within a novel bioreactor. The effect of mechanical loading and dexamethasone concentration on BMSC osteogenic differentiation and mineralized matrix production was studied for 8 and 16 days of culture. Mechanical stimulation after 16 days with 10 nM dexamethasone promoted osteogenic differentiation of BMSCs by significantly elevating alkaline phosphatase activity as well as alkaline phosphatase and osteopontin transcript levels over static controls. Mineralized matrix production also increased under these culture conditions. Dexamethasone concentration had a dramatic effect on the ability of mechanical stimulation to modulate these phenotypic and genotypic responses. These results provide increased insight into the role of mechanical stimulation on osteogenic differentiation of human BMSCs in vitro and may lead to improved strategies in bone tissue engineering.

Mesenchymal stem cells (MSCs) have been demonstrated as an attractive cell source for tissue-engineering applications because of their ability to be easily isolated and expanded from adult bone marrow aspirates and their versatility for pluripotent differentiation into mesenchymal tissues. This review highlights advances and progress in bone reconstruction techniques for both the repair of site-specific bone defects and the attenuation of musculoskeletal disease symptoms associated with osteoporosis and osteogenesis imperfecta. Despite the enormous potential benefits of MSCs within these approaches, conventional tissue culture methods limit the clinical utility of these cells because of the gradual loss of both their proliferative and differentiation potential during ex vivo expansion. Novel strategies to overcome these limitations are discussed including cultivation in the presence of basic fibroblastic growth factor 2, induction of ectopotic telomerase expression, and ex vivo expansion on various collagenous biomaterials. In addition, this review also outlines mechanistic theories on the potential role of MSC-extracellular matrix interactions in mediating the retention of MSC proliferative and differentiation capacity after ex vivo expansion on collagenous biomaterials.

The high frequency and mortality associated with breast cancer metastasis to bone has motivated efforts to elucidate tumor-stroma interactions in the bone microenvironment contributing to invasion and proliferation of metastatic cells. The development of engineered tissues has prompted the integration of engineered bone scaffolds into animal models as potential targets for metastatic spread. Silk scaffolds were coupled with bone morphogenetic protein-2 (BMP-2), seeded with bone marrow stromal cells (BMSC), and maintained in culture for 7 weeks, 4 weeks, and 1 day before s.c. implant in a mouse model of human breast cancer metastasis from the orthotopic site. Following injection of SUM1315 cells into mouse mammary fat pads, tumor burden of implanted tissues was observed only in 1-day scaffolds. Scaffold development and implantation was then reinitiated to identify the elements of the engineered bone that contribute to metastatic spread. Untreated scaffolds were compared with BMP-2-coupled, BMSC-seeded, or BMP-2/BMSC-combined treatment. Migration of SUM1315 cells was detected in four of four mice bearing scaffolds with BMP-2 treatment and with BMSC treatment, respectively, whereas only one of six mice of the BMP-2/BMSC combination showed evidence of metastatic spread. Histology confirmed active matrix modeling and stromal cell/fibroblast infiltration in scaffolds positive for the presence of metastasis. These results show the first successful integration of engineered tissues in a model system of human breast cancer metastasis. This novel platform now can be used in continued investigation of the bone environment and stem cell contributions to the process of breast cancer metastasis.

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