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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biomed Opt. Author manuscript; available in PMC 2010 September 1.
Published in final edited form as:
J Biomed Opt. 2009 Sep–Oct; 14(5): 050504.
doi:  10.1117/1.3233590
PMCID: PMC2778480
NIHMSID: NIHMS139820

Doppler optical micro-angiography improves the quantification of local fluid flow and shear stress within 3D porous constructs

Abstract

Traditional phase-resolved Doppler optical coherence tomography (PRDOCT) has been reported to have potential to characterize local fluid flow within microporous scaffold. In this work, we apply Doppler optical micro-angiography (DOMAG), a new imaging technique that was developed by combining optical micro-angiography (OMAG) with phase-resolved method, for improved assessment of local fluid flow and its derived parameters, shear stress and interconnectivity, within highly scattering porous constructs. Compared with PRDOCT, we demonstrate a dramatic improvement of DOMAG in quantifying flow-related properties within scaffolds in situ for functional tissue engineering.

Keywords: Tissue engineering, porous scaffold, fluid flow, shear stress, optical coherence tomography, optical micro-angiography

The aim of functional tissue engineering (FTE) is to grow a complete 3D bio-tissue coupled with appropriate, biological functionalities for implanting into the body with a purpose of fostering remodeling and regeneration of diseased tissue [1]. In order to provide a suitable template to support cell growth and to deliver sufficient amount of nutrients during tissue development, a 3D porous scaffold is one of critical ingredients involved in FTE. Its architectural properties, pore interconnectivity for example, largely determine the interactions among groups of cells, which facilitate cell-cell communications and tissue integrity [2]. A bioreactor is another key element in FTE providing the necessary physical regulatory environment to guide appropriate cell differentiation and tissue development. Mechanical stimulus such as fluid-induced shear stress has a well-known impact on cell morphology and orientation, and is also an important modulator of cell physiology. There is a considerable evidence that mechanical stress affects gene expression and subsequently, protein synthesis, cell proliferation and differentiation [3, 4], and significantly increase the biosynthetic activity in a range of different cell types [5, 6]. Whereas, in vitro studies have shown that mechanical stress in culture environment such as within bioreactors presents biphasic dose-response characteristics. This suggests that excessive stress force that produces cell distortion may also play a role to switch cells between motility and apoptosis programming [7]. To further understand the mechanisms of dynamic interaction between the cell growing behavior and the mechanical shear stress, a non-destructive imaging technique is needed that is capable of imaging the localized fluid flow and shear stress in situ within the entire porous scaffold (with a typical thickness of ~ millimeters), preferably at a level of the individual micro-pores. However, such requirement of monitoring and imaging in tissue engineering is difficult, if not impossible, to fulfill by use of currently available imaging technologies.

Recently, phase-resolved Doppler optical coherence tomography (PRDOCT) has been reported to have a potential to image local fluid flow, subsequently to characterize shear stress and pore-interconnectivity in 3D porous scaffolds [8]. Although promising, the performance of PRDOCT is severely limited by a background texture noise presented in the system, imposed by the optical heterogeneous property of the tissue sample [9]. Most recently, a novel imaging method, Doppler optical micro-angiography (DOMAG) is reported to evaluate the velocities of blood flow within microcirculatory tissue beds with much improved precision [10]. DOMAG is based on recently developed optical micro-angiography (OMAG) [10] that is capable of separating the optical signals backscattered by moving scatters from the optical signals backscattered by the static tissue background to provide a capability of imaging 3D flow, almost free of texture background noise. Combining with phase-resolved method developed in PRDOCT, DOMAG extracts flow velocities from OMAG flow signals. In this letter, we briefly discuss how DOMAG improves imaging fidelity of fluid flow by use of a flow phantom, and then we report the utility of DOMAG to explore fluid flow, shear stress and interconnectivity within 3D porous scaffolds with an unprecedented accuracy as compared to PRDOCT.

The configuration and operating principles of DOMAG system can be found elsewhere [10]. Briefly, the system used in this study employed a broadband infrared superluminescent diode with a central wavelength of 1300 nm. The spectral interferogram formed by lights between the sample and reference arms was sent to a home built high-speed spectrometer that employed a line scan infrared InGaAs detector to achieve an imaging speed of 20 frames per second (fps) with 1000 A scans (axial scans) in each B scan (lateral direction). The system has the imaging resolution of 16×16×8 μm3 at x-y-z direction, and an imaging depth of ~3 mm in air. The 3D imaging of tissue sample in situ was achieved by an X-Y galvanometer scanner that provided a scan range of 2.5 mm in both X (lateral) and Y (elevational) direction for this study.

To test DOMAG performance in imaging flow, we first used DOMAG to image a flow phantom. The phantom was made from gelatin mixed with 2% milk to simulate the heterogeneous tissue background within which a capillary tube with an inner diameter of ~200 μm was submerged and ~2% TiO2 particle solution was flowing in it. The Doppler angle was set at 85°. The flow rate was controlled by a precision syringe pump. Fig. 1(A) shows a cross-sectional OMAG structural image of the scanned flow phantom that is identical to the image obtained by frequency domain OCT (FDOCT). The phase difference result in Fig. 1(B) is described by conventional PRDOCT to represent the flow velocity information. Due to the optical heterogeneity of a static tissue background [denoted by Bt in Fig. 1(A)], a background noise [Nb in Fig. 1(B)] from the non-flow region of phantom was imposed onto the PRDOCT flow image, making it difficult for PRDOCT to precisely measure the small flow velocity [1113]. An additional problem in PRDOCT is the random noise [labeled with Nr in Fig. 1(B)] from the background with low backscattered signal, such as the air region in this phantom (labeled with Ba in A). Before evaluating flow signals, the segmentation method has to be used to extract the tissue regions of interest. These two types of artifacts from backgrounds are maximally suppressed with the advent of DOMAG imaging method. Figure 1(C) shows the corresponding OMAG flow image that delineates the scattering fluid flow with both background noise and random noise being rejected. OMAG method successfully separated the backscattering flow signals from the background signals, resulting in minimal noise production [11]. However, OMAG does not provide the velocity information of flowing regions. It seems straightforward to apply the phase-resolved technique to extract flow velocities from OMAG signals of flow. Unfortunately, the correlation condition between adjacent A scans required by the phase-resolved method is not achievable in OMAG because of Hilbert transformation used. In order to apply phase-resolved technique onto OMAG flow signals, DOMAG digitally reconstructed an ideal background which was totally homogeneous and reinforced a complete correlation between adjacent A scans. For instance, when homogeneous background was superimposed, the black region in Fig. 1(C) without correlation would acquire an ideal correlation. The conventional phase-resolved method could consequently be used on OMAG flow signals to evaluate the phase difference maps from which the velocity of flow signals can be generated. It is clear that DOMAG in Fig. 1(D) provides superior imaging performance due to the noise suppression in either tissue or air background when compared to Fig. 1(B). To better show the noise suppression by DOMAG, we extracted two signal profiles across the same depth position marked by red and blue lines in Fig. 1(B) and 1(D), respectively. The corresponding signal profiles are shown in Fig. 1(E). The phase differences (parabolic curve) in flow region are almost the same by different methods, but the background noise in DOMAG (~0.02 rad) is much smaller than that in PRDOCT (~0.15 rad), and the random noise in PRDOCT pointed by black arrow was removed in DOMAG due to its reconstruction procedure. Consequently, we expect that the phase SNR will be increased in DOMAG as compared to PRDOCT, delivering improved evaluation of fluid flow and shear stress.

Fig. 1
Typical cross-sectional images of the flow characteristics in a flow phantom. (A) OMAG structural image. Ba = background of air; Bt = background of tissue. (B) PRDOCT velocity image. Nr = random noise; Nb = background noise. (C) OMAG flow image. (D) DOMAG ...

Next, we applied DOMAG in non-invasive assessment of fluid flow through porous microstructures in chitosan scaffolds, widely used in FTE. Chitosan scaffolds with high porosity were fabricated as described in Ref. [8] that were then placed within a transparent sample chamber (ID=1.5mm) for DOMAG imaging. During imaging, the chamber was oriented to have a Doppler angle of 85 with respective to the incident probe beam. A precision syringe pump was used to deliver a constant input flow rate of 8 ml/hr. A 0.5% latex microsphere (0.3 μm in diameter) suspension was used as the light scattering medium. Shown in Fig. 2 are the representative results from a single B scan of the scaffold. Fig. 2(A) is the OMAG structural image taken before scattering medium flowed through the sample, from which the microstructures of pores are clearly delineated. Fig. 2(B) shows the corresponding OMAG image of localized fluid flow that permeates this cross-section shown in Fig. 2(A). However, this image only provides the backscattered signals (i.e. reflectance) from functional flow that does not indicate the flow velocity information, which is however needed for quantifying the localized shear stress. By applying DOMAG method, the phase difference map [Fig. 2(C)] as to the imaged fluid flow was extracted from Fig. 2(B), which could then be converted to the velocity values. Compared with the result from the same section obtained by PRDOCT [Fig. 2(D)], the fluid flow with relative small velocities corresponding to the small values of phase difference [15] can be successfully captured by DOMAG [Fig. 2(C)]. As discussed in our previous work, in PRDOCT background phase-noise is a barrier to image the detailed velocities of flow due to the heterogeneity of scaffold as seen in Fig. 2(D). When the scan rate was set at 20 kHz, the noise floor in PRDOCT was typically 0.4 rad for the scaffold studied, suggesting that PRDOCT might fail to measure flow velocities < 0.83mm/s. Therefore, slow flows (< 0.83mm/s) in the microstructures were masked due to the noise production in PRDOCT. Whereas, DOMAG is capable of reducing this noise level to 0.03 rad, which corresponds to the minimal detectable velocity of ~62 μm/s. As a consequence, the low fluid flow near the wall of micropores (>62 μm/s) will be detected by DOMAG. This advantage will make DOMAG a more powerful tool in imaging fluid flow in the cultured, complex micro-constructs that are usually perfused by a low input flow rate.

Fig. 2
In situ imaging results for a typical B scan of the porous scaffolds. (A) OMAG structural image taken before scattering medium flowed through the sample. And OMAG images of (B) fluid flows and (C) their velocities respectively. (D) corresponding PRDOCT ...

Lastly, we provide a comparison between these two techniques for measuring shear stress and interconnectivity through the use of the same data set scanned from a perfused scaffold. Fig. 3(A) and 3(B) show the directional phase difference maps generated by PRDOCT and DOMAG, respectively. It is noted that we have to reduce the noise artifacts in PRDOCT flow image in order to evaluate shear stress and pore interconnectivity from the flow images. As the noise level of DOMAG is low in the entire image, noise reduction is not required. Compared to the PRDOCT image in Fig. 3(A), Fig. 3(B) shows that DOMAG detects the fluid flow within almost all micropores in the scanned construct, and more importantly, it illustrates that the slow flows near the pore walls (e.g., pointed by black arrows) are detected by DOMAG that however are not visible by PRDOCT. The ability of DOMAG to image the flows near wall is important because they are critical to deduce the localized shear stresses [8]. These results indicate that DOMAG promises a non-destructive imaging technique to reliably determine the localized shear stress values within porous structures. The corresponding shear stresses are shown in Fig. 3(C) and 3(D), respectively. It can be seen that most values are distributed on the pore walls in Fig. 3(D) because slower flows emerging along walls were detected by DOMAG method. However, higher values were derived by PRDOCT [Fig. 3(C)] due to the high noise floor that had to be removed before calculating the shear stress values, leading to an overestimate of the localized shear stress. As a consequence, DOMAG is capable of more precisely detecting the shear stress distribution in perfused constructs. In Fig. 3(E), the probability of shear stress value shown by probability mass function (n=12) is compared between PRDOCT and DOMAG. Visually, the shear stresses via DOMAG were distributed less broadly than those via PRDOCT, indicating detectable shear stress became more uniform in DOMAG, which caused the mean shear stress to decrease. Furthermore, the pore-interconnectivity evaluation from the flow image maps from two methods are presented in Fig. 3(F) (n=12). The mean value of DMOAG is ~1.5 fold larger than that of PRDOCT, suggesting the advantage of DOMAG over PRDOCT in quantifying how much space being used for transfusion in the artificial tissues.

Fig. 3
In situ assessment of shear stress and interconnectivity in porous scaffold via DOMAG and PRDOCT, respectively. Shown are (A) PRDOCT and (B) DOMAG images of velocities of fluid flow, respectively; and the corresponding (C) PRDOCT and (D) DOMAG image of ...

In summary, we have successfully demonstrated the use of DOMAG for improved characterization of local fluid flow within highly scattering microporous scaffolds in situ. By rejecting the background noise imposed by the heterogeneous properties of tissue sample, OMAG is able to detect the slow fluid flow near the porous wall within the microporous tissue constructs. The improved assessment of shear stress and interconnectivity values based on the flow information shows promise for DOMAG to monitor the dynamic properties of an engineered tissue in a bioreactor.

Acknowledgments

The authors would like to thank Dr Pierre Bagnaninchi (Edinburgh University, UK) in assisting the preparation of porous chitosan scaffolds used in this study. The work was supported in part by research grants from the National Heart, Lung, and Blood Institute (R01 HL093140) and the American Heart Association (0855733G). The content is solely the responsibility of the authors and does not necessarily represent the official views of grant giving bodies.

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