p21-/- mice exhibit enhanced bone regeneration after injury.

BACKGROUND: p21(WAF1/CIP1/SDI1), a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(-/-) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling. METHODS: The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21-/- mouse model. p21-/- and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo µCT scanning. RESULTS: p21-/- mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the µCT analysis indicates that p21-/- mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively. CONCLUSIONS: Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.

Embryonic stem cells incorporate into newly formed bone and do not form tumors in an immunocompetent mouse fracture model.

Embryonic stem (ES) cells are a uniquely self-renewing, pluripotent population of cells that must be differentiated before being useful for cell therapy. Since most studies utilize subcutaneous implantation to test the in vivo functionality of ES cell-derived cells, the objective of the current study was to develop an appropriate and clinically relevant in vivo implantation system in which the behavior and tumorigenicity of ES cell-derived cells could be effectively tested in a tissue-specific (orthotopic) site. Male ES cells were differentiated either into osteoblasts or chondrocytes using protocols that were previously developed and published by our laboratory. The differentiated cells were implanted into a burr-hole fracture created in the proximal tibiae of immunocompetent female mice, strain matched to the ES cell line. The ability of the differentiated ES cell-derived cells (bearing the Y chromosome) to incorporate into the newly formed bone was assessed by micro-computed tomography imaging and histochemistry. ES cells differentiated with either osteogenic or chondrogenic medium supplementation formed a soft tissue mass that disrupted the normal bone architecture by 4 weeks after implantation in some mice. In contrast, mice receiving osteoblastic cells that were differentiated in a three-dimensional type 1 collagen gel showed evidence of new bone formation at the defect site without evidence of tumor formation for up to 8 weeks after implantation. In this injury model, type 1 collagen is more effective than medium supplementation at driving more complete differentiation of ES cells, as evidenced by reducing their tumorigenicity. Overall, the current study emphasizes the importance of using an appropriate orthotopic implantation system to effectively test the behavior and tumorigenicity of the cells in vivo.

Assessment of the efficacy of MRI for detection of changes in bone morphology in a mouse model of bone injury.

PURPOSE: To determine whether magnetic resonance imaging (MRI) could be used to track changes in skeletal morphology during bone healing using high-resolution micro-computed tomography (µCT) as a standard. We used a mouse model of bone injury to compare µCT with MRI. MATERIALS AND METHODS: Surgery was performed to induce a burr hole fracture in the mouse tibia. A selection of biomaterials was immediately implanted into the fractures. First we optimized the imaging sequences by testing different MRI pulse sequences. Then changes in bone morphology over the course of fracture repair were assessed using in vivo MRI and µCT. Histology was performed to validate the imaging outcomes. RESULTS: The rapid acquisition with relaxation enhancement (RARE) sequence provided sufficient contrast between bone and the surrounding tissues to clearly reveal the fracture. It allowed detection of the fracture clearly 1 and 14 days postsurgery and revealed soft tissue changes that were not clear on µCT. In MRI and µCT the fracture was seen at day 1 and partial healing was detected at day 14. CONCLUSION: The RARE sequence was the most suitable for MRI bone imaging. It enabled the detection of hard and even soft tissue changes. These findings suggest that MRI could be an effective imaging modality for assessing changes in bone morphology and pathobiology.

Collagen I scaffolds cross-linked with beta-glycerol phosphate induce osteogenic differentiation of embryonic stem cells in vitro and regulate their tumorigenic potential in vivo.

Embryonic stem cells (ESCs) have the potential to differentiate into all tissues of the adult organism. This, along with the ability for unlimited self-renewal, positions these cells for regenerative medicine approaches based on tissue engineering strategies. With the objective of developing a treatment regime for skeletal injuries and diseases, this study presents a novel protocol that effectively induces ESC differentiation into osteogenic and chondrogenic lineages while concurrently eliminating observed tumorigenicity during the period of observation after transplantation in vivo. Exposure to a collagen I matrix polymerized with beta-glycerol phosphate (BGP) induced the osteogenic differentiation of the ESCs with an efficiency of >80% without purification and/or lineage-specific cell selection. Furthermore, when the collagen I matrix was supplemented with chondroitin sulfate, chondrogenesis was promoted instead of osteogenesis. Interestingly, without purification of the differentiated cells from the collagen I matrix, these constructs did not lead to the formation of teratomas or tumors when implanted subcutaneously in a severe combined immunodeficiency (SCID). Furthermore, if undifferentiated ESCs were mixed with collagen I and then injected immediately (i.e., without previous in vitro differentiation), again, no teratomas or tumors were observed, whereas undifferentiated ESCs without collagen scaffolds all produced teratomas in this bioassay system. These results suggest that collagen I scaffolds not only induce osteogenic differentiation of ESCs, but also prevent ESCs from producing unwanted tumors when injected in vivo.

Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells.

BACKGROUND: The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined. METHODOLOGY/PRINCIPAL FINDINGS: P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-ß and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed. CONCLUSIONS/SIGNIFICANCE: Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular 'fate' decision making processes.

Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media.

The generation of tissue-engineered constructs from stem cells for the treatment of musculoskeletal diseases may have immense impact in regenerative medicine, but there are difficulties associated with stem cell culture and differentiation, including the use of serum. Here we present serum-free protocols for the successful production of murine embryonic stem cell (mESC) derived osteoblasts and chondrocytes on CultiSpher S macroporous microcarriers in stirred suspension bioreactors. Various inoculum forms and agitation rates were investigated. Produced osteogenic cells were implanted ectopically into SCID mice and orthotopically into a murine burr-hole fracture model. Osterix, osteocalcin and collagen type I were upregulated in osteogenic cultures, while aggrecan and collagen type II were upregulated in chondrogenic cultures. Histological analysis using alizarin red S, von Kossa and alcian blue staining confirmed the presence of osteoblasts and chondrocytes, respectively in cultured microcarriers and excised tissue. Finally, implantation of derived cells into a mouse fracture model revealed cellular integration without any tumor formation. Overall, microcarriers may provide a supportive scaffold for ESC expansion and differentiation in a serum-free bioprocess for in vivo implantation. These findings lay the groundwork for the development of clinical therapies for musculoskeletal injuries and diseases using hESCs and iPS cells.

Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors.

Since the derivation of human embryonic stem (hES) cells, their translation to clinical therapies has been met with several challenges, including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We, along with other groups, have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence, flow cytometry, and teratoma formation assays, we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly, the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices, which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies.

Reduced differentiation efficiency of murine embryonic stem cells in stirred suspension bioreactors.

The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of leukemia inhibitory factor (LIF). Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs.