no / This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300 μm ± 10–30 μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-β1 (p < 0.001), TGF-β2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-β combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2 + 3) and TGF-β(1 + 3) (p < 0.001). Any cell treated with TGF-β1, TGF-β(1 + 2) and TGF-β(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2 + 3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment. / Emailed Mansour for final draft 27/06/2016

Bone repair is modulated by different stimuli. There is evidence that the Transforming Growth Factor-beta (TGF-¿) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules also influence cell behaviour. This study aimed at determining the role of the TGF-¿s, Collagen type I, Fibronectin and Laminin in bone cell behaviour. To do this MG63 bone cells were used to examine cell adhesion and alignment to different micro-contact printed ECM protein patterns of different widths. The study also aimed at examining how TGF-¿1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell surface interactions, cell morphology, cell proliferation and integrin expression. Finally, this study also aimed at examining how the TGF-¿s and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-¿s influence ECM secretion and integrin expression. 5, 10, 25, 50 and 100¿m wide repeat gratings of Collagen type I, Fibronectin and Laminin patterns were stamp patterned onto glass slides and plated with MG63 cells at 50,000 cells per coverslip. Cells on the fibronectin pattern attached and elongated soon after seeding, but did not adhere readily to collagen and laminin and appeared more rounded until 18hrs after seeding. Cells aligned significantly well on the 50¿m and 100¿m wide fibronectin patterned coverslips with mean angles of alignment ~7.87¿ ¿ 3.06SD and 6.45¿ ¿ 5.08SD, respectively, compared to those with smaller width (p<0.001). In comparison, cells aligned less readily to the other two ECM proteins, showing optimal alignments of 9.66¿ ¿ 4.18SD and 14.36¿ ¿ 1.57SD to the 50¿m wide collagen and laminin patterns, respectively. Differences in cell length mirrored those of alignment, with cells acquiring the greatest length when showing the greatest degree of alignment. The results indicate that MG63 cells responded significantly better to 50 and 100¿m wide fibronectin patterns compared to those with smaller width (p<0.001) indicating that the cells may attach mostly via fibronectin specific integrins. Cell surface attachment was examined via a trypsinisation assay in which the time taken to trypsinise cells from the surface provided a means of assessing the strength of attachment. The results indicated that treatment with the solvent (HCl), TGF-¿1, 2 and 3 all decreased cell attachment, but this effect was significantly greater in the case of HCl and TGF-¿3 (p<0.001). However, there were significant differences in trypsinisation rates between HCl and TGF-¿3 (p<0.001). The wound healing response to the TGF-¿s and their solvent/carrier was also investigated in 300¿m ± 10-30¿m SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The results indicated that TGF-¿3 and HCl significantly enhance wound closure when compared against negative controls, TGF-¿1 and TGF-¿2 treatment (p<0.001). It was also found that TGF-¿1 and TGF-¿2 treatment significantly improved wound closure rate in comparison to the controls (p<0.001). Experiments were performed to determine if the HCl effects on wound closure were dose dependent. Cells were incubated with 20¿M, 40¿M, 80¿M and 160¿M concentrations of HCl prior to wounding and wound closure rates were recorded. Wound closure was dependent on HCl dose with the 80¿M and 160¿M concentrations inducing increases in wound closure rates that were both significantly greater than those induced by 20¿M, 40¿M and control treatments (p<0.001). However, there were significant differences in wound closure between the 80¿M and 160¿M treatment groups after 30hrs of treatment (p<0.001). The effect of different TGF-¿ isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. The results suggest that cell morphology changes were observed significantly more in cells treated with TGF-¿(2+3) and TGF-¿(1+3) (p<0.001). Any cell treated with TGF-¿1, TGF-¿(1+2) and TGF-¿(1+2+3) showed significantly less elongation compared to the control and other TGF-¿ isomers. In terms of proliferation rate, TGF-¿3 and TGF-¿(2+3) increased cell numbers more than TGF-¿1, TGF-¿2 and other combinations. TGF-¿1 and its combinations did not show significant proliferation and attachment compared to the control due to perhaps its inhibitory effect in contact with human bone cells. Immunostaining indicated that treatment with TGF-¿3 significantly promoted the secretion of collagen type I and anti-human fibronectin in addition to integrin (¿3 and ¿1) expression. Statistically TGF-¿3 and their combinations showed significant differences in number of cells stained for collagen type I, anti-human fibronectin, ¿3 and ¿1integrin. Any cell treated with TGF-¿1 or any combination with TGF-¿1 showed significantly lower cell number stained with the same proteins and integrins (p<0.001). Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. WSPR images revealed guided cells with high contrast band like structures at the border of cells distal to the edge of guidance cue to which they aligned and with less concentrically formed band like features across the cell body. It is believed that the high contrast features are associated with the formation of focal contacts on the edge of the cells distal to the edge of fibronectin patterns, which suggests that cell guidance is aided by a decrease in cell attachment along a guidance feature. The WSPR experiments also indicated that TGF-¿s influenced the distribution of focal contacts. In the case of TGF-¿1 treated cells the bright high contrast regions were intense but only arranged around the periphery of the cell. In TGF-¿2 and TGF-¿3 cells the bright contrast regions were weaker but again mostly localised around the periphery. These findings supported the earlier trypsinisation results.

Bone repair is modulated by different stimuli. There is evidence that the Transforming Growth Factor-beta (TGF-β) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules also influence cell behaviour. This study aimed at determining the role of the TGF-βs, Collagen type I, Fibronectin and Laminin in bone cell behaviour. To do this MG63 bone cells were used to examine cell adhesion and alignment to different micro-contact printed ECM protein patterns of different widths. The study also aimed at examining how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell surface interactions, cell morphology, cell proliferation and integrin expression. Finally, this study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence ECM secretion and integrin expression. 5, 10, 25, 50 and 100μm wide repeat gratings of Collagen type I, Fibronectin and Laminin patterns were stamp patterned onto glass slides and plated with MG63 cells at 50,000 cells per coverslip. Cells on the fibronectin pattern attached and elongated soon after seeding, but did not adhere readily to collagen and laminin and appeared more rounded until 18hrs after seeding. Cells aligned significantly well on the 50μm and 100μm wide fibronectin patterned coverslips with mean angles of alignment ~7.87° ± 3.06SD and 6.45° ± 5.08SD, respectively, compared to those with smaller width (p<0.001). In comparison, cells aligned less readily to the other two ECM proteins, showing optimal alignments of 9.66° ± 4.18SD and 14.36° ± 1.57SD to the 50μm wide collagen and laminin patterns, respectively. Differences in cell length mirrored those of alignment, with cells acquiring the greatest length when showing the greatest degree of alignment. The results indicate that MG63 cells responded significantly better to 50 and 100μm wide fibronectin patterns compared to those with smaller width (p<0.001) indicating that the cells may attach mostly via fibronectin specific integrins. Cell surface attachment was examined via a trypsinisation assay in which the time taken to trypsinise cells from the surface provided a means of assessing the strength of attachment. The results indicated that treatment with the solvent (HCl), TGF-β1, 2 and 3 all decreased cell attachment, but this effect was significantly greater in the case of HCl and TGF-β3 (p<0.001). However, there were significant differences in trypsinisation rates between HCl and TGF-β3 (p<0.001). The wound healing response to the TGF-βs and their solvent/carrier was also investigated in 300μm ± 10-30μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The results indicated that TGF-β3 and HCl significantly enhance wound closure when compared against negative controls, TGF-β1 and TGF-β2 treatment (p<0.001). It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p<0.001). Experiments were performed to determine if the HCl effects on wound closure were dose dependent. Cells were incubated with 20μM, 40μM, 80μM and 160μM concentrations of HCl prior to wounding and wound closure rates were recorded. Wound closure was dependent on HCl dose with the 80μM and 160μM concentrations inducing increases in wound closure rates that were both significantly greater than those induced by 20μM, 40μM and control treatments (p<0.001). However, there were significant differences in wound closure between the 80μM and 160μM treatment groups after 30hrs of treatment (p<0.001). The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. The results suggest that cell morphology changes were observed significantly more in cells treated with TGF-β(2+3) and TGF-β(1+3) (p<0.001). Any cell treated with TGF-β1, TGF-β(1+2) and TGF-β(1+2+3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2+3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control due to perhaps its inhibitory effect in contact with human bone cells. Immunostaining indicated that treatment with TGF-β3 significantly promoted the secretion of collagen type I and anti-human fibronectin in addition to integrin (α3 and β1) expression. Statistically TGF-β3 and their combinations showed significant differences in number of cells stained for collagen type I, anti-human fibronectin, α3 and β1 integrin. Any cell treated with TGF-β1 or any combination with TGF-β1 showed significantly lower cell number stained with the same proteins and integrins (p<0.001). Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. WSPR images revealed guided cells with high contrast band like structures at the border of cells distal to the edge of guidance cue to which they aligned and with less concentrically formed band like features across the cell body. It is believed that the high contrast features are associated with the formation of focal contacts on the edge of the cells distal to the edge of fibronectin patterns, which suggests that cell guidance is aided by a decrease in cell attachment along a guidance feature. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In the case of TGF-β1 treated cells the bright high contrast regions were intense but only arranged around the periphery of the cell. In TGF-β2 and TGF-β3 cells the bright contrast regions were weaker but again mostly localised around the periphery. These findings supported the earlier trypsinisation results.