Novel diagnostic tools with the ability to monitor variations in biochemical composition and provide benchmark indicators of vascular tissue maturation are needed to create functional tissue replacements. We investigated the ability of fiber-based, label-free multispectral fluorescent lifetime imaging (FLIm) to quantify the anatomical variations in biochemical composition of native carotid arteries and validated these results against biochemical assays. FLIm-derived parameters in spectral band 415-455 nm correlated with tissue collagen content (R² = 0.64) and cell number (R² = 0.61) and in spectral band 465-553 nm strongly correlated with elastin content (R² = 0.89). These results suggest that FLIm holds great potential for assessing vascular tissue maturation and functional properties based on tissue autofluorescence.

Significance

Cartilage tissue engineering is a promising strategy for effective curative therapies for treatment of osteoarthritis. However, tissue engineers depend predominantly on time-consuming, expensive, and destructive techniques as quality control to monitor the maturation of engineered cartilage. This practice can be impractical for large-scale biomanufacturing and prevents spatial and temporal monitoring of tissue growth, which is critical for the fabrication of clinically relevant-sized cartilage constructs. Nondestructive multimodal imaging techniques combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) hold great potential to address this challenge.

Aim

The feasibility of using multimodal FLIm-OCT for nondestructive, spatial, and temporal monitoring of self-assembled cartilage tissue maturation in a preclinical mouse model is investigated.

Approach

Self-assembled cartilage constructs were developed for 4 weeks in vitro followed by 4 weeks of in vivo maturation in nude mice. Sterile and nondestructive in situ multispectral FLIm and OCT imaging were carried out at multiple time points ( t=2 , 4, and 8 weeks) during tissue development. FLIm and 3D volumetric OCT images were reconstructed and used for the analysis of tissue biochemical homogeneity, morphology, and structural integrity. A biochemical homogeneity index was computed to characterize nonhomogeneous tissue growth at different time points. OCT images were validated against histology.

Results

FLIm detects heterogenous extracellular matrix (ECM) growth of tissue-engineered cartilage. The outer edge of the tissue construct exhibited longer fluorescence lifetime in 375 to 410 and 450 to 485 nm spectral channels, indicating increase in collagen content. Significant ( p<0.05 ) decrease of construct homogeneity index was observed between t=2 weeks and t=4 weeks. Both FLIm and OCT images revealed defects (voids) at the center of the tissue construct during in vitro culture ( t=2 and 4 weeks). Cyst formation during in vivo culture was detected by OCT and confirmed with histology.

Conclusions

The ability of multimodal FLIm-OCT to nondestructively monitor the heterogenous growth of engineered tissue constructs in situ is demonstrated. Spatial and temporal variation of construct ECM component was detected by FLIm. OCT reveals structural defects (voids and cysts). This multimodal approach has great potential to replace costly destructive tests in the manufacturing of tissue-engineered medical products, facilitating their clinical translation.

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

Glycosaminoglycan (GAG) loss is an early marker of osteoarthritis, which is a clinical late stage disease that affects millions of people worldwide. The goal of our study was to evaluate the ability of a fiber-based fluorescence lifetime imaging (FLIm) technique to detect GAG loss in articular cartilage. Native bovine cartilage explants (n  =  20) were exposed to 0 (control), 0.5 (low), or 1  U  /  mL (high) concentrations of chondroitinase ABC (cABC) to create samples with different levels of GAG loss. FLIm assessment (excitation: 355 nm; detection: channel 1: 375 to 410 nm, channel 2: 450 to 485 nm, channel 3: 530 to 565 nm) was conducted on depth-resolved cross-sections of the cartilage sample. FLIm images, validated with histology, revealed that loss of GAG resulted in a decrease of fluorescence lifetime values in channel 2 (Δ  =  0.44  ns, p  <  0.05) and channel 3 (Δ  =  0.75  ns, p  <  0.01) compared to control samples (channel 2: 6.34 ns; channel 3: 5.22 ns). Fluorescence intensity ratio values were lower in channel 1 (37%, p  <  0.0001) and channel 2 (31% decrease, p  <  0.0001) and higher in channel 3 (23%, p  <  0.0001) relative to control samples. These results show that FLIm can detect the loss of GAG in articular cartilage and support further investigation into the feasibility of in vivo FLIm arthroscopy.

Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition, microstructure, and function of engineered tissue equivalents. A non-destructive solution to monitor tissue quality and maturation would greatly reduce costs and accelerate the development of tissue-engineered products. The objectives of this study were to (a) determine whether matrix stabilization with exogenous lysyl oxidase-like protein-2 (LOXL2) with recombinant hyaluronan and proteoglycan link protein-1 (LINK) would result in increased compressive and tensile properties in self-assembled articular cartilage constructs, (b) evaluate whether label-free, non-destructive fluorescence lifetime imaging (FLIm) could be used to infer changes in both biochemical composition and biomechanical properties, (c) form quantitative relationships between destructive and non-destructive measurements to determine whether the strength of these correlations is sufficient to replace destructive testing methods, and (d) determine whether support vector machine (SVM) learning can predict LOXL2-induced collagen crosslinking. The combination of exogenous LOXL2 and LINK proteins created a synergistic 4.9-fold increase in collagen crosslinking density and an 8.3-fold increase in tensile strength as compared with control (CTL). Compressive relaxation modulus was increased 5.9-fold with addition of LOXL2 and 3.4-fold with combined treatments over CTL. FLIm parameters had strong and significant correlations with tensile properties (R²  = 0.82; p < 0.001) and compressive properties (R²  = 0.59; p < 0.001). SVM learning based on FLIm-derived parameters was capable of automating tissue maturation assessment with a discriminant ability of 98.4%. These results showed marked improvements in mechanical properties with matrix stabilization and suggest that FLIm-based tools have great potential for the non-destructive assessment of tissue-engineered cartilage.

There is a critical need to develop noninvasive, nondestructive methods for assessing the quality of engineered constructs prior to implantation. Currently, the composition and maturity of engineered tissues are assessed using destructive, costly, and time-consuming biochemical and mechanical analyses. The goal of this study was to use noninvasive, multimodal imaging to monitor osteogenic differentiation and matrix deposition by human mesenchymal stem/stromal cells (MSCs) during in vitro culture. MSCs were encapsulated in alginate hydrogels and cultured in osteogenic conditions for 4 weeks. Samples were evaluated using fluorescence lifetime imaging (FLIm) and ultrasound backscatter microscopy (UBM) prior to traditional biochemical and mechanical testing. Using linear regression analysis, we identified strong correlations between imaging parameters (e.g., fluorescence lifetime and acoustic attenuation coefficient) and destructive mechanical and biochemical tests to assess the maturation of osteogenically induced constructs. These data demonstrate the promise of nondestructive label-free imaging techniques to noninvasively ascertain the progression and maturity of tissue engineered bone grafts.

Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecan-coated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-β1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-β1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.

Regulatory guidelines for tissue engineered products require stringent characterization during production and necessitate the development of novel, non-destructive methods to quantify key functional parameters for clinical translation. Traditional assessments of engineered tissues are destructive, expensive, and time consuming. Here, we introduce a non-destructive, inexpensive, and rapid sampling and analysis system that can continuously monitor the mechanical, biochemical, and structural properties of a single sample over extended periods of time. The label-free system combines the imaging modalities of fluorescent lifetime imaging and ultrasound backscatter microscopy through a fiber-based interface for sterile monitoring of tissue quality. We tested the multimodal system using tissue engineered articular cartilage as an experimental model. We identified strong correlations between optical and destructive testing. Combining FLIm and UBM results, we created a novel statistical model of tissue homogeneity that can be applied to tissue engineered constructs prior to implantation. Continuous monitoring of engineered tissues with this non-destructive system has the potential for in-process monitoring of tissue engineered products, reducing costs and improving quality controls in research, manufacturing, and clinical applications.

Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were observed in the host. Addition of aggregate redifferentiation culture prior to construct formation improved chondrogenic differentiation of foreskin-derived hDIAS cells, as evident by increases in glycosaminoglycan and collagen contents. More robust Alcian blue staining and homogeneous cell populations were also observed compared to controls. Human DIAS cells elicited no adverse host responses, reacted positively to chondrogenic regimens, and possessed multi-lineage differentiation potential with the caveat that efficacy may differ by anatomical origin of the skin. Taken together, these results suggest that hDIAS cells hold promise as a potential cell source for a number of tissue engineering applications.

Bovine pericardium (BP) is a vascular biomaterial used in cardiovascular surgery that is typically cross-linked for masking antigenicity and enhance stability. There is a need for biochemical evaluation of the tissue properties prior to implantation to ensure that quality and reliability standards are met. Here, engineered antigen removed BP (ARBP) that was cross-linked with 0.2% and 0.6% glutaraldehyde (GA), and further calcified in vitro to simulate graft calcifications upon implantation was characterized nondestructively using fluorescence lifetime imaging (FLIm) to identify regions of interest which were then assessed by Raman spectroscopy. We observed that the tissue fluorescence lifetime shortened, and that Raman bands at 856, 935, 1282, and 1682 cm^-1 decreased, and at 1032 and 1627 cm^-1 increased with increasing GA cross-linking. Independent classification analysis based on fluorescence lifetime and on Raman spectra discriminated between GA-ARBP and untreated ARBP with an accuracy of 91% and 66%, respectively. Pearson's correlation analysis showed a strong correlation between pyridinium cross-links measured with high-performance liquid chromatography and fluorescence lifetime measured at 380-400 nm (R = -0.76, p = 0.00094), as well as Raman bands at 856 cm^-1 for hydroxy-proline (R = -0.68, p = 0.0056) and at 1032 cm^-1 for hydroxy-pyridinium (R = 0.74, p = 0.0016). Calcified areas of GA cross-linked tissue showed characteristic hydroxyapatite (959 and 1038 cm^-1) bands in the Raman spectrum and fluorescence lifetime shortened by 0.4 ns compared to uncalcified regions. FLIm-guided Raman imaging could rapidly identify degrees of cross-linking and detected calcified regions with high chemical specificity, an ability that can be used to monitor tissue engineering processes for applications in regenerative medicine.