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Salter's type I11 and type IV growth plate injuries often induce bone bridge formation at the injury site. To understand the cellular mechanisms, this study characterized proximal tibia1 transphyseal injury in rats. Histologically, bony bridge trabeculae appeared on day 7, increased on day 10, and became well-constructed on day 14 with marrow. Prior to and during bone bridging, there was no cartilage proteoglycan metachromatic staining and no collagen-)< immunostaining at the injury site, nor was there any up-regulation of BrdU-labelled chondrocyte proliferation at the adjacent physeal cartilage, suggesting no new cartilage formation at the injury site. However, infiltration of vimentin-immunopositive mesenchymal cells from metaphysis and epiphysis was apparent on day 3, with the mesenchymal population being prominent on days 7 and 10 and subsided on day 14. Among these infiltrates were osteoprogenitor precursors expressing osteoblast differentiation factor (cbf-al ) on day 3, along with some cbf-a1 osteoblast-like cells lining bone trabeculae on days 7 and 10. Some mesenchymal cells and trabecula-lining cells were also alkaline phosphataseimmunopositive, further suggesting their osteoblast differentiation. From day 7 onwards, some trabecula-lining cells became osteocalcin-producing mature osteoblasts. These results suggest that bone bridge formation after growth plate injury occurs directly via intramembranous ossification through recruitment of marrow-derived osteoprogenitor cells.Crown

Since discovery, significant interest has been generated in the potential application of mesenchymal stem cells or multipotential stromal cells (MSC) for tissue regeneration and repair, due to their proliferative and multipotential capabilities. Although the sheep is often used as a large animal model for translating potential therapies for musculoskeletal injury and repair, the characteristics of MSC from ovine bone marrow have been inadequately described. Histological and gene expression studies have previously shown that ovine MSC share similar properties with human and rodents MSC, including their capacity for clonogenic growth and multiple stromal lineage differentiation. In the present study, ovine bone marrow derived MSCs positively express cell surface markers associated with MSC such as CD29, CD44 and CD166, and lacked expression of CD14, CD31 and CD45. Under serum-deprived conditions, proliferation of MSC occurred in response to EGF, PDGF, FGF-2, IGF-1 and most significantly TGF-alpha. While subcutaneous transplantation of ovine MSC in association with a ceramic HA/TCP carrier into immunocomprimised mice resulted in ectopic osteogenesis, adipogenesis and haematopoietic-support activity, transplantation of these cells within a gelatin sponge displayed partial chondrogenesis. The comprehensive characterisation of ovine MSC described herein provides important information for future translational studies involving ovine MSC.

Intensive use of cancer chemotherapy is increasingly linked with long-term skeletal side effects such as osteopenia, osteoporosis and fractures. However, cellular mechanisms by which chemotherapy affects bone integrity remain unclear. Methotrexate (MTX), used commonly as an anti-metabolite, is known to cause bone defects. To study the pathophysiology of MTX-induced bone loss, we examined effects on bone and marrow fat volume, population size and differentiation potential of bone marrow stromal cells (BMSC) in adult rats following chemotherapy for a short-term (five once-daily doses at 0.75 mg/kg) or a 6-week term (5 doses at 0.65 mg/kg + 9 days rest + 1.3 mg/kg twice weekly for 4 weeks). Histological analyses revealed that both acute and chronic MTX treatments caused a significant decrease in metaphyseal trabecular bone volume and an increase in marrow adipose mass. In the acute model, proliferation of BMSCs significantly decreased on days 3-9, and consistently the stromal progenitor cell population as assessed by CFU-F formation was significantly reduced on day 9. Ex vivo differentiation assays showed that while the osteogenic potential of isolated BMSCs was significantly reduced, their adipogenic capacity was markedly increased on day 9. Consistently, RT-PCR gene expression analyses showed osteogenic transcription factors Runx2 and Osterix (Osx) to be decreased but adipogenic genes PPARγ and FABP4 up-regulated on days 6 and 9 in the stromal population. These findings indicate that MTX chemotherapy reduces the bone marrow stromal progenitor cell population and induces a switch in differentiation potential towards adipogenesis at the expense of osteogenesis, resulting in osteopenia and marrow adiposity.

Wnt signalling has an essential role in regulating bone formation and remodelling during embryonic development and throughout postnatal and adult life. Specifically, Wnt signalling regulates bone formation by controlling embryonic cartilage development and postnatal chondrogenesis, osteoblastogenesis, osteoclastogenesis, endochondral bone formation, and bone remodelling. Abnormalities in the function of Wnt genes give rise to or contribute to the development of several pathological bone conditions, including abnormal bone mass, osteosarcomas and bone loss in multiple myeloma. Furthermore, Wnt signalling is activated during bone fracture repair and plays a crucial role in regulating bone regeneration.

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Injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Previously, injury-induced, initial inflammatory response was shown to be an acute inflammatory event containing predominantly neutrophils. To examine potential roles of neutrophils in the bony repair, a neutrophil-neutralizing antiserum or control normal serum was administered systemically in rats with growth plate injury. The inflammatory response was found temporally associated with increased expression of neutrophil chemotactic chemokine cytokine-induced neutrophil chemoattractant-1 and cytokines TNF-alpha and IL-1beta. Following the inflammatory response, mesenchymal infiltration, chondrogenic and osteogenic responses, and bony repair were observed at the injury site. Neutrophil reduction did not significantly affect infiltration of other inflammatory cells and expression of TNF-alpha and IL-1beta and growth factors, platelet-derived growth factor-B and TGF-beta1, at the injured growth plate on Day 1 and had no effects on mesenchymal infiltration on Day 4. By Day 10, however, there was a significant reduction in proportion of mesenchymal repair tissue but an increase (although statistically insignificant) in bony trabeculae and a decrease in cartilaginous tissue within the injury site. Consistently, in antiserum-treated rats, there was an increase in expression of osteoblastic differentiation transcription factor cbf-alpha1 and bone matrix protein osteocalcin and a decrease in chondrogenic transcription factor Sox-9 and cartilage matrix collagen-II in the injured growth plate. These results suggest that injury-induced, neutrophil-mediated inflammatory response appears to suppress mesenchymal cell osteoblastic differentiation but enhance chondrogenic differentiation, and thus, it may be involved in regulating downstream chondrogenic and osteogenic events for growth plate bony repair.

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