Application of OCT-Derived Attenuation Coefficient in Acute Burn-Damaged Skin.

BACKGROUND AND OBJECTIVES: There remains a need to objectively monitor burn wound healing within a clinical setting, and optical coherence tomography (OCT) is proving itself one of the ideal modalities for just such a use. The aim of this study is to utilize the noninvasive and multipurpose capabilities of OCT, along with its cellular-level resolution, to demonstrate the application of optical attenuation coefficient (OAC), as derived from OCT data, to facilitate the automatic digital segmentation of the epidermis from scan images and to work as an objective indicator for burn wound healing assessment. STUDY DESIGN/MATERIALS AND METHODS: A simple, yet efficient, method was used to estimate OAC from OCT images taken over multiple time points following acute burn injury. This method enhanced dermal-epidermal junction (DEJ) contrast, which facilitated the automatic segmentation of the epidermis for subsequent thickness measurements. In addition, we also measured and compared the average OAC of the dermis within said burns for correlative purposes. RESULTS: Compared with unaltered OCT maps, enhanced DEJ contrast was shown in OAC maps, both from single A-lines and completed B-frames. En face epidermal thickness and dermal OAC maps both demonstrated significant changes between imaging sessions following burn injury, such as a loss of epidermal texture and decreased OAC. Quantitative analysis also showed that OAC of acute burned skin decreased below that of healthy skin following injury. CONCLUSIONS: Our study has demonstrated that the OAC estimated from OCT data can be used to enhance imaging contrast to facilitate the automatic segmentation of the epidermal layer, as well as help elucidate our understanding of the pathological changes that occur in human skin when exposed to acute burn injury, which could serve as an objective indicator of skin injury and healing.

Imaging human skin autograft integration with optical coherence tomography.

BACKGROUND: Skin autografting is a common clinical procedure for reconstructive surgery. Despite its widespread use, very few studies have been conducted to non-invasively evaluate and monitor the vascular and structural features of skin grafts. This study, therefore, aims to demonstrate the potential of optical coherence tomography (OCT) alongside OCT-based angiography (OCTA) to non-invasively image and monitor human skin graft health and integration over time. METHODS: An in-house-built clinical prototype OCT system was used to acquire OCT/OCTA images from patients who underwent split-thickness skin graft surgery following severe burn damage to the skin. The OCT imaging was carried out at multiple locations over multiple time points with a field of view of ~9 mm × 9 mm and a penetration depth of ~1.5 mm. In addition to obtaining high-resolution qualitative images, we also quantitatively measured and compared specific structural and vascular parameters, such as identifiable layer thickness and corresponding vascular area density and diameter. RESULTS: Two patients (patient #1 and #2) were enrolled for this preliminary study. Vascular and structural features were successfully imaged and measured in the graft tissue and integration layer immediately beneath at different time points. Revascularization, healing, and integration were monitored with patient-specific details. Results of the quantitative image analysis from patient #1 indicated that integration layer thickness 16-day post-surgery was significantly less (~50%) than that of 7-day post-surgery. Additionally, with patient #2, significant growth (~20%) was seen with the vascular area density of both the graft and corresponding integration layer beneath between 6 and 14 days post-surgery. CONCLUSIONS: Our preliminary studies show that OCT/OCTA has clinical potential to image and measure numerous features of human skin graft health and integration in the days and weeks following split-thickness surgery. For the first time, we demonstrate the applicability of non-invasive imaging technology for novel clinical uses that could eventually aid in the betterment of surgical practices and clinical outcomes.

OCT-Based Angiography and Surface Topography in Burn-Damaged Skin.

BACKGROUND AND OBJECTIVES: There is a clinical need for an accurate, non-invasive imaging tool that can provide the objective assessment of burn wounds. The aims of this study are to demonstrate the potential of optical coherence tomography (OCT) in evaluating burn wound healing, as well as exploring the physiological basis of human wound healing. STUDY DESIGN/MATERIALS AND METHODS: This was a retrospective study. Seven patients with severe burn wounds who were admitted to Harborview Medical Center were imaged using an in-house-built, clinical-prototype OCT system. OCT imaging was carried out at multiple scan sites on the burned skin across two time points (imaging session #1 and #2) with a field of view of ~9 × 9 mm. Due to pathological differences among burn zones, scan sites were classified into red sites (zone of hyperemia), white sites (zone of coagulation), and mixed sites. In addition to obtaining qualitative en face vascular and surface topography maps, we quantified vascular area density and surface roughness for comparative purposes. RESULTS: En face vascular and surface topography maps demonstrated numerous morphological changes over both imaging sessions associated with burn injury, such as altered blood flow and loss of regular texture. Quantitative analyses revealed that during imaging session #1, vascular area density was significantly increased within the red sites compared with that of a healthy control (P = 0.0130), while vascular area density was significantly decreased within the white sites compared with that of a healthy control (P < 0.0001). During imaging session #2, vascular area density was significantly reduced to a more normal range within the red sites compared with imaging session #1 (P = 0.0215); however, vascular area density was still significantly lower within the white sites compared with that of a healthy control (P < 0.0001). Furthermore, vascular area density and surface roughness were significantly increased within the white sites during imaging session #2 compared with imaging session #1 (both P < 0.0001). CONCLUSIONS: OCT is clinically feasible to monitor vascular changes and alterations in skin surface roughness during the process of burn wound healing. Variations in vascular area density and roughness measurements within the burn wounds revealed by OCT offer some key insights into the underlying pathophysiological mechanisms responsible for wound healing, which may become critical biological indicators in future clinical evaluation and monitoring of wound healing. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Regulation of endothelial cell arrangements within hMSC - HUVEC co-cultured aggregates.

BACKGROUND: Micro-mass culturing or cellular aggregation is an effective method used to form mineralised bone tissue. Poor core cell viability, however, is often an impeding characteristic of large micro-mass cultures, and equally for large tissue-engineered bone grafts. Because of this, efforts are being made to enhance large graft perfusion, often through pre-vascularisation, which involves the co-culture of endothelial cells and bone cells or stem cells. METHODS: This study investigated the effects of different aggregation techniques and culture conditions on endothelial cell arrangements in mesenchymal stem cell and human umbilical vein endothelial cell co-cultured aggregates when endothelial cells constituted just 5%. Two different cellular aggregation techniques, i.e. suspension culture aggregation and pellet culture aggregation, were applied alongside two subsequent culturing techniques, i.e. hydrostatic loading and static culturing. Endothelial cell arrangements were assessed under such conditions to indicate potential pre-vascularisation. RESULTS: Our study found that the suspension culture aggregates cultured under hydrostatic loading offered the best environment for enhanced endothelial cell regional arrangements, closely followed by the pellet culture aggregates cultured under hydrostatic loading, the suspension culture aggregates cultured under static conditions, and the pellet culture aggregates cultured under static conditions. CONCLUSIONS: The combination of particular aggregation techniques with dynamic culturing conditions appeared to have a synergistic effect on the cellular arrangements within the co-cultured aggregates.

Optical coherence tomography correlates multiple measures of tissue damage following acute burn injury.

BACKGROUND: The visual assessment of burned skin is inherently subjective, and whilst a number of imaging modalities have identified quantifiable parameters to characterize vascular and structural changes following burn damage, none have become common place in the assessment protocol. Here, we use optical coherence tomography (OCT)-based angiography (OCTA) to introduce novel correlations between vessel depth, i.e., the depth of functional blood vessels beneath the tissue surface, edema depth, i.e., the depth of interstitial fluid buildup beneath the tissue surface, and tissue injury depth, i.e., the depth of collagen denaturation beneath the tissue surface, following burn injury. METHODS: A clinical prototype OCT system was used to collect OCT images from various sites of burned skin in patients. Optical microangiography (OMAG) algorithm was used to derive OCTA information from the acquired OCT images, from which the presence of blood vessels and edema were detected. The optical attenuation mapping of structural OCT information was used to detect tissue injury depth. The depths of vessel, edema and tissue injury were measured using a semi-automatic segmentation algorithm. Correlation analysis was performed using a Pearson correlation coefficient using one-tailed analysis with significance being established by a P value ≤0.05. RESULTS: Four burn patients were recruited and scanned at multiple sites using the prototype system within 3-6 days of injury. Approximate measurements include a vessel depth range of 320-1,360 µm, an edema depth range of 0-400 µm, and a tissue injury depth range of 130-420 µm. Correlations were subsequently observed between vessel depth and edema depth (r=0.8521, P=0.0001), and vessel depth and tissue injury depth (r=0.6296, P=0.0106). CONCLUSIONS: OCT is feasible to provide the critical information of vessel depth, edema depth, and tissue injury depth of skin burns, which may represent viable assessment criteria for the characterization of cutaneous burns in future.

Microvascular imaging of the skin.

Despite our understanding that the microvasculature plays a multifaceted role in the development and progression of various conditions, we know little about the extent of this involvement. A need exists for non-invasive, clinically meaningful imaging modalities capable of elucidating microvascular information to aid in our understanding of disease, and to aid in the diagnosis/monitoring of disease for more patient-specific care. In this review article, a number of imaging techniques are summarized that have been utilized to investigate the microvasculature of skin, along with their advantages, disadvantages and future perspectives in preclinical and clinical settings. These techniques include dermoscopy, capillaroscopy, Doppler sonography, laser Doppler flowmetry (LDF) and perfusion imaging, laser speckle contrast imaging (LSCI), optical coherence tomography (OCT), including its Doppler and dynamic variant and the more recently developed OCT angiography (OCTA), photoacoustic imaging, and spatial frequency domain imaging (SFDI). Attention is largely, but not exclusively, placed on optical imaging modalities that use intrinsic optical signals to contrast the microvasculature. We conclude that whilst each imaging modality has been successful in filling a particular niche, there is no one, all-encompassing modality without inherent flaws. Therefore, the future of cutaneous microvascular imaging may lie in utilizing a multi-modal approach that will counter the disadvantages of individual systems to synergistically augment our imaging capabilities.

Spatial and Temporal Heterogeneities of Capillary Hemodynamics and Its Functional Coupling During Neural Activation.

The cerebral vascular system provides a means to meet the constant metabolic needs of neuronal activities in the brain. Within the cerebral capillary bed, the interactions of spatial and temporal hemodynamics play a deterministic role in oxygen diffusion, however, the progression of which remains unclear. Taking the advantages of high-spatiotemporal resolution of optical coherence tomography capillary velocimetry designed with the eigen-decomposition statistical analysis, we investigated intrinsic red blood cell (RBC) velocities and their spatiotemporal adjustment within the capillaries permeating mouse cerebral cortex during electrical stimulation of contralateral hind paw. We found that the mean capillary transit velocity (mCTV) is increased and its temporal fluctuation bandwidth (TFB) is broadened within hind-paw somatosensory cortex. In addition, the degree to which the mCTV is increased negatively correlates with resting state mCTV, and the degree to which the TFB is increased negatively correlates with both the resting state mCTV and the TFB. In order to confirm the changes are due to hemodynamic regulation, we performed angiographic analyses and found that the vessel density remains almost constant, suggesting the observed functional activation does not involve recruitment of reserved capillaries. To further differentiate the contributions of the mCTV and the TFB to the spatiotemporally coupled hemodynamics, changes in the mCTV and TBF of the capillary flow were modeled and investigated through a Monte Carlo simulation. The results suggest that neural activation evokes the spatial transit time homogenization within the capillary bed, which is regulated via both the heterogeneous acceleration of RBC flow and the heterogeneous increase of temporal RBC fluctuation, ensuring sufficient oxygenation during functional hyperemia.

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds.

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.

Comparing imaging capabilities of spectral domain and swept source optical coherence tomography angiography in healthy subjects and central serous retinopathy.

BACKGROUND: There are two forms of system implementation of optical coherence tomography angiography (OCTA) in ophthalmic imaging, i.e., spectral domain (SD-) and swept source OCTA (SS-OCTA). The purpose of this paper is to compare the SD-OCTA and SS-OCTA for elucidating structural and vascular features associated with central serous retinopathy (CSR), and to evaluate the effects of CSR on SD- and SS-OCTA's imaging capabilities. METHODS: Normal subjects and CSR patients were imaged by SD- and SS-OCTA using 3 × 3 mm and 6 × 6 mm scan patterns. OCT signal strengths at the superficial retina, deep retina, Sattler's layer and Haller's layer were used to compare the ability of SD- and SS-OCTA to image structural features. In addition, the ability to acquire angiograms were discussed by evaluating retinal vessel density. Central serous volume (CSV) was measured and it was correlated with difference in signal strengths (∆S) between two OCTA devices. RESULTS: Seven normal eyes and seven diseased eyes were recruited. Results showed no significant differences between SD- and SS-OCT in detecting structural features of the retinal layer according to the paired t-test. However, when imaging the Sattler's layer for normal eyes, a significant difference is found between SD- and SS-OCT (p < 0.0001 for 3 × 3 mm scan, and p = 0.0002 for 6 × 6 mm); while for CSR eyes, the corresponding values were p < 0.0001 and p = 0.0003, respectively. At Haller's layer for normal eyes, the corresponding values were p = 0.0004 and p = 0.0014; and for CSR eyes, p = 0.0004 and p < 0.0001, respectively. A strong correlation between ∆S and CSV was observed in the Sattler's layer (3 × 3 mm - p = 0.0031 and R2  = 0.951; 6 × 6 mm - p = 0.0075 and R2  = 0.911) and Haller's layer (3 × 3 mm - p = 0.0026 and R2  = 0.955; 6 × 6 mm - p = 0.0013 and R2  = 0.972). CONCLUSIONS: The results suggest no differences between SD- and SS-OCTA for imaging the retinal layers however, when imaging beyond retinal layers, SS-OCTA appears advantageous in detecting returning signals. In CSR cases, the CSV may have an impact on sub-CSR tissue imaging and appears to have more impact on SD- than SS-OCTA.

OCT-based angiography of human dermal microvascular reactions to local stimuli: Implications for increasing capillary blood collection volumes.

OBJECTIVES: To measure and compare microvascular responses within the skin of the upper arm to local stimuli, such as heating or rubbing, through the use of optical coherence tomography angiography (OCTA), and to investigate its impact on blood volume collection. MATERIALS AND METHODS: With the use of heat packs or rubbing, local stimulation was applied to the skin of either the left or right upper arm. Data from the stimulated sites were obtained using OCTA comparing pre- and post-stimulation microvascular parameters, such as vessel density, mean vessel diameter, and mean avascular pore size. Additionally, blood was collected using a newly designed collection device and volume was recorded to evaluate the effect of the skin stimulation. RESULTS: Nineteen subjects were recruited for local stimulation study (including rubbing and heating) and 21 subjects for blood drawn study. Of these subjects, 14 agreed to participate in both studies. OCTA was successful in monitoring and measuring minute changes in the microvasculature of the stimulated skin. Compared to baseline, significant changes after local heating and rubbing were respectively found in vessel density (16% [P = 0.0004] and 33% [P < 0.0001] increase), mean vessel diameter (14% and 11% increase) and mean avascular pore size (5% [P = 0.0068] and 8% [P = 0.0005] decrease) after stimulations. A gradual recovery was recorded for each parameter, with no difference being measured after 30 minutes. Blood collection volumes significantly increased after stimulations of heating (48% increase; P = 0.049) and rubbing (78% increase; P = 0.048). Significant correlations were found between blood volume and microvascular parameters except mean avascular pore size under the heating condition. CONCLUSIONS: OCTA can provide important information regarding microvascular adaptations to local stimuli. With that, both heating and rubbing of the skin have positive effects on blood collection capacity, with rubbing having the most significant effect. Lasers Surg. Med. 50:908-916, 2018. © 2018 Wiley Periodicals, Inc.

Optical coherence tomography angiography monitors human cutaneous wound healing over time.

BACKGROUND: In vivo imaging of the complex cascade of events known to be pivotal elements in the healing of cutaneous wounds is a difficult but essential task. Current techniques are highly invasive, or lack the level of vascular and structural detail required for accurate evaluation, monitoring and treatment. We aimed to use an advanced optical coherence tomography (OCT)-based angiography (OCTA) technique for the non-invasive, high resolution imaging of cutaneous wound healing. METHODS: We used a clinical prototype OCTA to image, identify and track key vascular and structural adaptations known to occur throughout the healing process. Specific vascular parameters, such as diameter and density, were measured to aid our interpretations under a spatiotemporal framework. RESULTS: We identified multiple distinct, yet overlapping stages, hemostasis, inflammation, proliferation, and remodeling, and demonstrated the detailed vascularization and anatomical attributes underlying the multifactorial processes of dermatologic wound healing. CONCLUSIONS: OCTA provides an opportunity to both qualitatively and quantitatively assess the vascular response to acute cutaneous damage and in the future, may help to ascertain wound severity and possible healing outcomes; thus, enabling more effective treatment options.

Optical coherence tomography angiography of normal skin and inflammatory dermatologic conditions.

BACKGROUND: In clinical dermatology, the identification of subsurface vascular and structural features known to be associated with numerous cutaneous pathologies remains challenging without the use of invasive diagnostic tools. OBJECTIVE: To present an advanced optical coherence tomography angiography (OCTA) method to directly visualize capillary-level vascular and structural features within skin in vivo. METHODS: An advanced OCTA system with a 1310 nm wavelength was used to image the microvascular and structural features of various skin conditions. Subjects were enrolled and OCTA imaging was performed with a field of view of approximately 10 × 10 mm. Skin blood flow was identified using an optical microangiography (OMAG) algorithm. Depth-resolved microvascular networks and structural features were derived from segmented volume scans, representing tissue slabs of 0-132, 132-330, and 330-924 µm, measured from the surface of the skin. RESULTS: Subjects with both healthy and pathological conditions, such as benign skin lesions, psoriasis, chronic graft-versus-host-disease (cGvHD), and scleroderma, were OCTA scanned. Our OCTA results detailed variations in vascularization and local anatomical characteristics, for example, depth-dependent vascular, and structural alterations in psoriatic skin, alongside their resolve over time; vascular density changes and distribution irregularities, together with corresponding structural depositions in the skin of cGvHD patients; and vascular abnormalities in the nail folds of a patient with scleroderma. CONCLUSION: OCTA can image capillary blood flow and structural features within skin in vivo, which has the potential to provide new insights into the pathophysiology, as well as dynamic changes of skin diseases, valuable for diagnoses, and non-invasive monitoring of disease progression and treatment. Lasers Surg. Med. 50:183-193, 2018. © 2018 Wiley Periodicals, Inc.

Current State-of-the-Art 3D Tissue Models and Their Compatibility with Live Cell Imaging.

Mammalian cells grow within a complex three-dimensional (3D) microenvironment where multiple cells are organized and surrounded by extracellular matrix (ECM). The quantity and types of ECM components, alongside cell-to-cell and cell-to-matrix interactions dictate cellular differentiation, proliferation and function in vivo. To mimic natural cellular activities, various 3D tissue culture models have been established to replace conventional two dimensional (2D) culture environments. Allowing for both characterization and visualization of cellular activities within possibly bulky 3D tissue models presents considerable challenges due to the increased thickness and subsequent light scattering features of such 3D models. In this chapter, state-of-the-art methodologies used to establish 3D tissue models are discussed, first with a focus on both scaffold-free and scaffold-based 3D tissue model formation. Following on, multiple 3D live cell imaging systems, mainly optical imaging modalities, are introduced. Their advantages and disadvantages are discussed, with the aim of stimulating more research in this highly demanding research area.

Automatic motion correction for in vivo human skin optical coherence tomography angiography through combined rigid and nonrigid registration.

When using optical coherence tomography angiography (OCTA), the development of artifacts due to involuntary movements can severely compromise the visualization and subsequent quantitation of tissue microvasculatures. To correct such an occurrence, we propose a motion compensation method to eliminate artifacts from human skin OCTA by means of step-by-step rigid affine registration, rigid subpixel registration, and nonrigid B-spline registration. To accommodate this remedial process, OCTA is conducted using two matching all-depth volume scans. Affine transformation is first performed on the large vessels of the deep reticular dermis, and then the resulting affine parameters are applied to all-depth vasculatures with a further subpixel registration to refine the alignment between superficial smaller vessels. Finally, the coregistration of both volumes is carried out to result in the final artifact-free composite image via an algorithm based upon cubic B-spline free-form deformation. We demonstrate that the proposed method can provide a considerable improvement to the final en face OCTA images with substantial artifact removal. In addition, the correlation coefficients and peak signal-to-noise ratios of the corrected images are evaluated and compared with those of the original images, further validating the effectiveness of the proposed method. We expect that the proposed method can be useful in improving qualitative and quantitative assessment of the OCTA images of scanned tissue beds.

Influence of Additive Manufactured Scaffold Architecture on the Distribution of Surface Strains and Fluid Flow Shear Stresses and Expected Osteochondral Cell Differentiation.

Scaffolds for regenerative medicine applications should instruct cells with the appropriate signals, including biophysical stimuli such as stress and strain, to form the desired tissue. Apart from that, scaffolds, especially for load-bearing applications, should be capable of providing mechanical stability. Since both scaffold strength and stress-strain distributions throughout the scaffold depend on the scaffold's internal architecture, it is important to understand how changes in architecture influence these parameters. In this study, four scaffold designs with different architectures were produced using additive manufacturing. The designs varied in fiber orientation, while fiber diameter, spacing, and layer height remained constant. Based on micro-CT (µCT) scans, finite element models (FEMs) were derived for finite element analysis (FEA) and computational fluid dynamics (CFD). FEA of scaffold compression was validated using µCT scan data of compressed scaffolds. Results of the FEA and CFD showed a significant impact of scaffold architecture on fluid shear stress and mechanical strain distribution. The average fluid shear stress ranged from 3.6 mPa for a 0/90 architecture to 6.8 mPa for a 0/90 offset architecture, and the surface shear strain from 0.0096 for a 0/90 offset architecture to 0.0214 for a 0/90 architecture. This subsequently resulted in variations of the predicted cell differentiation stimulus values on the scaffold surface. Fluid shear stress was mainly influenced by pore shape and size, while mechanical strain distribution depended mainly on the presence or absence of supportive columns in the scaffold architecture. Together, these results corroborate that scaffold architecture can be exploited to design scaffolds with regions that guide specific tissue development under compression and perfusion. In conjunction with optimization of stimulation regimes during bioreactor cultures, scaffold architecture optimization can be used to improve scaffold design for tissue engineering purposes.

Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM.

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone.

A facile in vitro model to study rapid mineralization in bone tissues.

BACKGROUND: Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required. METHODS: We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT). RESULTS: Two size aggregates (on average, large aggregates were 745 µm and small 79 µm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture. CONCLUSIONS: This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.