Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
Ultrasound Med Biol. 2011 Oct;37(10):1734-42. doi: 10.1016/j.ultrasmedbio.2011.06.010. Epub 2011 Aug 25.
Acoustic microscopy was used to monitor an ex vivo produced oral mucosal equivalent (EVPOME) developed on acellular cadaveric dermis (AlloDerm®). As seeded cells adhered and grew, they filled in and smoothed out the surface irregularities, followed by the production of a keratinized protective outermost layer. If noninvasive in vitro ultrasonic monitoring of these cellular changes could be developed, then tissue cultivation could be adjusted in-process to account for biologic variations in the development of these stratified cell layers. Cultured keratinocytes (from freshly obtained oral mucosa) were harvested and seeded onto AlloDerm® coated with human type IV collagen and cultured 11 days. EVPOMEs were imaged on the 11th day post-seeding using a scanning acoustic microscope (SAM) that consists of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f-number. The specimen surface was determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise: 20-40 dB above the signal for the water and 2-dimensional (2-D) ultrasonic images were created using confocal image reconstruction. A known area from each micrograph was divided into 12-40 even segments and examined for surface irregularities. These irregularities were quantified and one-way analysis of variance (ANOVA) and linear regression analysis were performed to correlate the surface profiles for both the AlloDerm® and EVPOME specimens imaged by SAM. Histology micrographs of the AlloDerm® and EVPOME specimens were also prepared and examined for surface irregularities. Unseeded AlloDerm® averaged seven to nine surface changes per 400 μm. The number of changes in surface irregularities decreased to two to three per 400 μm on the mature EVPOMEs. The numbers of surface irregularities between the unseeded AlloDerm® vs. developing EVPOME are similar for both histology and SAM 2-D B-scan images. For the EVPOME 2-D B-scan micrographs produced by SAM, the decrease in surface irregularities is indicative of the stratified epithelium formed by seeded oral keratinocytes; verified in the histology images between the AlloDerm® and EVPOME. A near 1:1 linear correlation shows the similarities between the two imaging modalities. SAM demonstrates its ability to discern the cell development and differentiation occurring on the EVPOME devices. Unlike histology, SAM measurements are noninvasive and can be used to monitor tissue graft development without damaging any cells/tissues.
声显微镜被用于监测在脱细胞尸体真皮（AlloDerm®）上体外培养的口腔黏膜等效物（EVPOME）。随着接种细胞的附着和生长，它们填补并平滑了表面的不规则处，随后产生了一层角质化的保护性最外层。如果可以开发出对这些细胞变化的非侵入性体外超声监测，那么可以在培养过程中调整组织培养，以适应这些分层细胞层发育中的生物学变化。从新鲜获得的口腔黏膜中收获培养的角质形成细胞，并接种到涂有人 IV 型胶原蛋白的 AlloDerm®上，培养 11 天。在接种后第 11 天，使用扫描声学显微镜（SAM）对 EVPOME 进行成像，SAM 由单个元件换能器组成：61 MHz 中心频率，32 MHz 带宽，1.52 f-number。通过将信号的幅度阈值设定为安全高于噪声的第一个轴向入射值来确定标本表面：水的信号为 20-40 dB 以上，二维（2-D）超声图像使用共焦图像重建创建。从每个显微照片的已知区域中划分 12-40 个均匀的片段，并检查表面不规则性。对这些不规则性进行量化，并进行单因素方差分析（ANOVA）和线性回归分析，以相关 SAM 成像的 AlloDerm®和 EVPOME 标本的表面轮廓。还制备了 AlloDerm®和 EVPOME 标本的组织学显微照片，并检查了表面不规则性。未接种的 AlloDerm®平均每 400 μm 有 7-9 个表面变化。在成熟的 EVPOME 上，表面不规则性的变化数量减少到每 400 μm 有 2-3 个。未接种的 AlloDerm®与发育中的 EVPOME 之间的表面不规则性数量相似，这两种方法的组织学和 SAM 2-D B 扫描图像均如此。对于 SAM 产生的 EVPOME 2-D B 扫描显微照片，表面不规则性的减少表明由接种的口腔角质形成细胞形成的分层上皮；在 AlloDerm®和 EVPOME 之间的组织学图像中得到证实。几乎是 1:1 的线性相关性表明了两种成像方式的相似性。SAM 证明了它能够分辨 EVPOME 装置上发生的细胞发育和分化。与组织学不同，SAM 测量是非侵入性的，可以用于监测组织移植物的发育，而不会损坏任何细胞/组织。
