Ament Jared D, Spurr-Michaud Sandra J, Dohlman Claes H, Gipson Ilene K
Massachusetts Eye and Ear Infirmary; Harvard Medical School, Boston, Massachusetts, USA.
Cornea. 2009 Aug;28(7):808-11. doi: 10.1097/ICO.0b013e31819670ac.
To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro).
Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P < 0.05 was statistically significant.
HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36).
This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated.
在体外确定用于波士顿人工角膜(KPro)时,钛在角膜细胞相容性方面是否优于标准聚甲基丙烯酸甲酯(PMMA)。
将人角膜缘上皮(HCLE)细胞单独培养在培养板中(作为对照),或与PMMA或钛盘共同培养24、48、72、96、120、144或168小时。实验重复进行三次(最终n = 6)。为了确定材料是否释放出可溶性毒性因子,在48小时和144小时时进行了同步实验,将圆盘置于Transwell支持物中,在其下方接种HCLE细胞。作为对可溶性因子的额外测试,将细胞与圆盘条件培养基孵育24小时,并在每个时间点通过增殖测定法对每孔活细胞数量进行定量。为了确定细胞增殖延迟是否归因于细胞死亡,在所有条件下测量HCLE细胞死亡情况,并在每个时间点通过细胞毒性测定法进行定量。通过重复测量方差分析确定材料随时间对HCLE细胞增殖的影响。P < 0.05具有统计学意义。
与PMMA相比,含有钛盘的孔中HCLE细胞增殖更多。随着时间的推移,测试圆盘与对照非圆盘共培养物之间在细胞增殖(P = 0.001)和死亡(P = 0.0025)方面的差异具有统计学意义。使用Transwells(P = 0.9836)或圆盘条件培养基(P = 0.36)未发现显著差异。
该体外HCLE细胞模型表明,与细胞/PMMA接触相比,细胞/钛接触时细胞增殖显著增加,细胞死亡减少。此外,差异不太可能归因于可溶性因子。表明需要对这两种KPro生物材料进行前瞻性体内分析。