In Situ Assessment of Porcine Osteochondral Repair Tissue in the Visible-Near Infrared Spectral Region.

Standard assessment of cartilage repair progression by visual arthroscopy can be subjective and may result in suboptimal evaluation. Visible-near infrared (Vis-NIR) fiber optic spectroscopy of joint tissues, including articular cartilage and subchondral bone, provides an objective approach for quantitative assessment of tissue composition. Here, we applied this technique in the 350-2,500 nm spectral region to identify spectral markers of osteochondral tissue during repair with the overarching goal of developing a new approach to monitor repair of cartilage defects in vivo. Full thickness chondral defects were created in Yucatan minipigs using a 5-mm biopsy punch, and microfracture (MFx) was performed as a standard technique to facilitate repair. Tissues were evaluated at 1 month (in adult pigs) and 3 months (in juvenile pigs) post-surgery by spectroscopy and histology. After euthanasia, Vis-NIR spectra were collected in situ from the defect region. Additional spectroscopy experiments were carried out in vitro to aid in spectral interpretation. Osteochondral tissues were dissected from the joint and evaluated using the conventional International Cartilage Repair Society (ICRS) II histological scoring system, which showed lower scores for the 1-month than the 3-month repair tissues. In the visible spectral region, hemoglobin absorbances at 540 and 570 nm were significantly higher in spectra from 1-month repair tissue than 3-month repair tissue, indicating a reduction of blood in the more mature repair tissue. In the NIR region, we observed qualitative differences between the two groups in spectra taken from the defect, but differences did not reach significance. Furthermore, spectral data also indicated that the hydrated environment of the joint tissue may interfere with evaluation of tissue water absorbances in the NIR region. Together, these data provide support for further investigation of the visible spectral region for assessment of longitudinal repair of cartilage defects, which would enable assessment during routine arthroscopy, particularly in a hydrated environment.

Assessment of tissue damage from mosquito-inspired surgical needle.

INTRODUCTION: Many percutaneous procedures utilize surgical needles to extract tissue samples in biopsy or to apply specific cancer treatments. A design of mosquito-inspired surgical needles was proposed to improve the efficacy of these procedures by reducing the needle insertion force and the resulting tissue damage. The focus of this study is to assess tissue damage caused by the insertion of a mosquito-inspired needle into soft tissues. MATERIAL AND METHODS: In this work, the geometric features and the dynamic stinging (insertion) mechanism of mosquito proboscis were mimicked for the design of 3D-manufactured bioinspired needle prototypes. A specially designed test setup was developed to measure the insertion force in bovine liver tissue. The histology assessment based on hematoxylin and eosin staining and image analysis was conducted to determine the bovine liver tissue damage. RESULTS: It was observed that the insertion force can be reduced by up to 39% and the bovine liver tissue damage was decreased by 27% using the mosquito-inspired needles when compared with using the standard needles. CONCLUSION: The findings from this study suggested that the bioinspired needle design has great potential to advance surgical needles for more effective and less invasive percutaneous procedures.

Nondestructive assessment of tissue engineered cartilage based on biochemical markers in cell culture media: application of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy.

Tissue engineering of cartilage for tissue repair has many challenges, including the inability to assess when the developing construct has reached compositional maturity for implantation. The goal of this study was to provide a novel analytical approach to nondestructively assess tissue engineered cartilage (TEC) during in vitro development. We applied attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to establish a quick and straightforward method to evaluate consumption of glucose and secretion of the metabolite lactate in the culture media, processes that are associated with tissue development. Using a series of standards, we showed by principal component analysis (PCA) that ATR-FTIR data was able to distinguish culture media with varying amounts of glucose and lactate. The 2nd derivative spectra displayed specific peaks of glucose at 1035 cm-1 and lactate at 1122 cm-1, and both the spectral first principal component (PC-1) scores and the 1122/1035 peak ratio very strongly correlated with the concentration of these components. TEC was prepared using chondrogenic cells grown in hydrogels, and analyzed for cell viability, distribution, and formation of proteoglycan (PG, a major cartilage protein). ATR-FTIR data of the cell culture media harvested during TEC development showed that the spectral PC-1 and the 1122/1035 peak ratio could significantly distinguish cultures with different amounts of constructs (1, 3 or 5 constructs per well) or with constructs at different developmental stages (3 or 5 weeks of culture). Interestingly, we also found that the PG content of the TEC was significantly correlated with both spectral PC-1 (r = -0.79) and the 1122/1035 peak ratio (r = 0.80). Therefore, monitoring relative glucose and lactate concentrations in cell culture media by ATR-FTIR provides a novel nondestructive approach to assess development of TEC.

Applications of Vibrational Spectroscopy for Analysis of Connective Tissues.

Advances in vibrational spectroscopy have propelled new insights into the molecular composition and structure of biological tissues. In this review, we discuss common modalities and techniques of vibrational spectroscopy, and present key examples to illustrate how they have been applied to enrich the assessment of connective tissues. In particular, we focus on applications of Fourier transform infrared (FTIR), near infrared (NIR) and Raman spectroscopy to assess cartilage and bone properties. We present strengths and limitations of each approach and discuss how the combination of spectrometers with microscopes (hyperspectral imaging) and fiber optic probes have greatly advanced their biomedical applications. We show how these modalities may be used to evaluate virtually any type of sample (ex vivo, in situ or in vivo) and how "spectral fingerprints" can be interpreted to quantify outcomes related to tissue composition and quality. We highlight the unparalleled advantage of vibrational spectroscopy as a label-free and often nondestructive approach to assess properties of the extracellular matrix (ECM) associated with normal, developing, aging, pathological and treated tissues. We believe this review will assist readers not only in better understanding applications of FTIR, NIR and Raman spectroscopy, but also in implementing these approaches for their own research projects.

Characterization of connective tissues using near-infrared spectroscopy and imaging.

Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no risk of laser-induced photothermal damage. A typical NIR spectroscopy workflow for biological tissue characterization involves sample preparation, spectral acquisition, pre-processing and analysis. The resulting spectrum embeds intrinsic information on the tissue's biomolecular, structural and functional properties. Here we demonstrate the analytical power of NIR spectroscopy for exploratory and diagnostic applications by providing instructions for acquiring NIR spectra, maps and images in biological tissues. By adapting and extending this protocol from the demonstrated application in connective tissues to other biological tissues, we expect that a typical NIR spectroscopic study can be performed by a non-specialist user to characterize biological tissues in basic research or clinical settings. We also describe how to use this protocol for exploratory study on connective tissues, including differentiating among ligament types, non-destructively monitoring changes in matrix formation during engineered cartilage development, mapping articular cartilage proteoglycan content across bovine patella and spectral imaging across the depth-wise zones of articular cartilage and subchondral bone. Depending on acquisition mode and experiment objectives, a typical exploratory study can be completed within 6 h, including sample preparation and data analysis.

DMOG Negatively Impacts Tissue Engineered Cartilage Development.

OBJECTIVE: Articular cartilage exists in a hypoxic environment, which motivates the use of hypoxia-simulating chemical agents to improve matrix production in cartilage tissue engineering. The aim of this study was to investigate whether dimethyloxalylglycine (DMOG), a HIF-1α stabilizer, would improve matrix production in 3-dimensional (3D) porcine synovial-derived mesenchymal stem cell (SYN-MSC) co-culture with chondrocytes. DESIGN: Pellet cultures and scaffold-based engineered cartilage were grown in vitro to determine the impact of chemically simulated hypoxia on 2 types of 3D cell culture. DMOG-treated groups were exposed to DMOG from day 14 to day 21 and grown up to 6 weeks with n = 3 per condition and time point. RESULTS: The addition of DMOG resulted in HIF-1α stabilization in the exterior of the engineered constructs, which resulted in increased regional type II collagen deposition, but the stabilization did not translate to overall increased extracellular matrix deposition. There was no increase in HIF-1α stabilization in the pellet cultures. DMOG treatment also negatively affected the mechanical competency of the engineered cartilage. CONCLUSIONS: Despite previous studies that demonstrated the efficacy of DMOG, here, short-term treatment with DMOG did not have a uniformly positive impact on the chondrogenic capacity of SYN-MSCs in either pellet culture or in scaffold-based engineered cartilage, as evidenced by reduced matrix production. Such 3D constructs generally have a naturally occurring hypoxic center, which allows for the stabilization of HIF-1α in the interior tissue. Thus, short-term addition of DMOG may not further improve this in cartilage tissue engineered constructs.

Approaches for In Situ Monitoring of Matrix Development in Hydrogel-Based Engineered Cartilage.

Near infrared (NIR) spectroscopy using a fiber optic probe shows great promise for the nondestructive in situ monitoring of tissue engineered construct development; however, the NIR evaluation of matrix components in samples with high water content is challenging, as water absorbances overwhelm the spectra. In this study, we established approaches by which NIR spectroscopy can be used to select optimal individual engineered hydrogel constructs based on matrix content and mechanical properties. NIR spectroscopy of dry standard compounds allowed identification of several absorbances related to collagen and/or proteoglycan (PG), of which only two could be identified in spectra obtained from hydrated constructs, at ∼5940 and 5800 cm-1. In dry sample mixtures, the ratio of these peaks correlated positively to collagen and negatively to PG. In NIR spectra from engineered cartilage hydrogels, these peaks reflected higher collagen and PG content and dynamic modulus values, permitting the differentiation of constructs with poor and good matrix development. Similarly, the increasing baseline offset in raw NIR spectra also reflected matrix development in hydrated constructs. However, weekly monitoring of NIR spectra and the peaks at ∼5940 and 5800 cm-1 was not adequate to differentiate individual constructs based on matrix composition. Interestingly, changes in the baseline offset of raw spectra could be used to evaluate the growth trajectory of individual constructs. These results demonstrate an optimal approach for the use of fiber optic NIR spectroscopy for in situ monitoring of the development of engineered cartilage, which will aid in identifying individual constructs for implantation. Impact statement A current demand in tissue engineering is the establishment of nondestructive approaches to evaluate construct development during growth in vitro. In this article, we demonstrate original nondestructive approaches by which fiber optic NIR spectroscopy can be used to assess matrix (PG and collagen) formation and mechanical properties in hydrogel-based constructs. Our data provide a cohesive molecular-based approach for in situ longitudinal evaluation of construct development during growth in vitro. The establishment of these approaches is a valuable step toward the real-time identification and selection of constructs with optimal properties, which may lead to successful tissue integration upon in vivo implantation.

Spatial correlation of native and engineered cartilage components at micron resolution.

Tissue engineering (TE) approaches are being widely investigated for repair of focal defects in articular cartilage. However, the amount and/or type of extracellular matrix (ECM) produced in engineered constructs does not always correlate with the resultant mechanical properties. This could be related to the specifics of ECM distribution throughout the construct. Here, we present data on the amount and distribution of the primary components of native and engineered cartilage (i.e., collagen, proteoglycan (PG), and water) using Fourier transform infrared imaging spectroscopy (FT-IRIS). These data permit visualization of matrix and water at 25 µm resolution throughout the tissues, and subsequent colocalization of these components using image processing methods. Native and engineered cartilage were cryosectioned at 80 µm for evaluation by FT-IRIS in the mid-infrared (MIR) and near-infrared (NIR) regions. PG distribution correlated strongly with water in native and engineered cartilage, supporting the binding of water to PG in both tissues. In addition, NIR-derived matrix peaks correlated significantly with MIR-derived collagen peaks, confirming the interpretation that these absorbances arise primarily from collagen and not PG. The combined use of MIR and NIR permits assessment of ECM and water spatial distribution at the micron level, which may aid in improved development of TE techniques.

Infrared Spectroscopic Quantification of Methacrylation of Hyaluronic Acid: A Scaffold for Tissue Engineering Applications.

Methacrylated hyaluronic acid (MeHA) has been used extensively in tissue engineering and drug delivery applications. The degree of methacrylation (DM) of HA impacts hydrogel crosslinking, which is of pivotal importance for cell interactions. The methacrylation reaction occurs over several hours, and DM is currently assessed post reaction and after dialysis of the solution, using nuclear magnetic resonance (1H NMR) data. Thus, there is little control over exact DM in a specific reaction. Here, infrared (IR) spectroscopy in attenuated total reflection (ATR) mode was investigated as an alternate modality for assessment of the DM of HA hydrogels, including during the reaction progression. Attenuated total reflection is a low-cost technique that is widely available in research and industry labs that can be used online during the reaction process. Strong correlations were achieved with IR-derived peak heights from dialyzed and lyophilized samples at 1708 cm-1 (from the methacrylic ester carbonyl vibration), and 1H NMR values ( R = 0.92, P = 6.56E-11). Additional IR peaks of importance were identified using principal component analysis and resulted in significant correlations with the 1H NMR DM parameter: 1454 cm-1 ( R = 0.85, P = 2.81E-8), 1300 cm-1 ( R = 0.95, P = 4.50E-14), 950 ( R = 0.85, P = 3.55E-8), 856 cm-1 ( R = 0.94, P = 1.20E-12), and 809 cm-1 ( R = 0.93, P = 3.54E-12). A multiple linear regression model to predict 1H NMR-derived DM using the 1708, 1300, and 1200 cm-1 peak heights as independent variables resulted in prediction with an error of 3.2% using dialyzed and lyophilized samples ( P < 0.001). Additionally, a multilinear regression model to predict the DM in undialyzed liquid MeHA samples obtained during the reaction process using similar peak height positions as independent variables resulted in a prediction error of 0.81% ( P < 0.05). Thus, IR spectroscopy can be utilized as an alternate modality to 1H NMR for quantification of the DM of MeHA while sampling either on-line during the methacrylation reaction as well as in post-lyophilized products. This could greatly simplify workflow for tissue engineering and other applications.

Vibrational spectroscopy and imaging: applications for tissue engineering.

Tissue engineering (TE) approaches strive to regenerate or replace an organ or tissue. The successful development and subsequent integration of a TE construct is contingent on a series of in vitro and in vivo events that result in an optimal construct for implantation. Current widely used methods for evaluation of constructs are incapable of providing an accurate compositional assessment without destruction of the construct. In this review, we discuss the contributions of vibrational spectroscopic assessment for evaluation of tissue engineered construct composition, both during development and post-implantation. Fourier transform infrared (FTIR) spectroscopy in the mid and near-infrared range, as well as Raman spectroscopy, are intrinsically label free, can be non-destructive, and provide specific information on the chemical composition of tissues. Overall, we examine the contribution that vibrational spectroscopy via fiber optics and imaging have to tissue engineering approaches.

Induction of endometrial mesenchymal stem cells into tissue-forming cells suitable for fascial repair.

Pelvic organ prolapse is a major hidden burden affecting almost one in four women. It is treated by reconstructive surgery, often augmented with synthetic mesh. To overcome the growing concerns of using current synthetic meshes coupled with the high risk of reoperation, a tissue engineering strategy has been developed, adopting a novel source of mesenchymal stem cells. These cells are derived from the highly regenerative endometrial lining of the uterus (eMSCs) and will be delivered in vivo using a new gelatin-coated polyamide scaffold. In this study, gelatin properties were optimized by altering the gelatin concentration and extent of crosslinking to produce the desired gelation and degradation rate in culture. Following cell seeding of uncoated polyamide (PA) and gelatin-coated meshes (PA+G), the growth rate of eMSCs on the PA+G scaffolds was more than that on the PA alone, without compromising cell shape. eMSCs cultured on the PA+G scaffold retained their phenotype, as demonstrated by W5C5/SUSD2 (eMSC-specific marker) immunocytochemistry. Additionally, eMSCs were induced to differentiate into smooth muscle cells (SMC), as shown by immunofluorescence for smooth muscle protein 22 and smooth muscle myosin heavy chain. eMSCs also differentiated into fibroblast-like cells when treated with connective tissue growth factor with enhanced detection of Tenascin-C and collagen type I as well as new tissue formation, as seen by Masson's trichrome. In summary, it was demonstrated that the PA+G scaffold is an appropriate platform for eMSC delivery, proliferation and differentiation into SMC and fibroblasts, with good biocompatibility and the capacity to regenerate neo-tissue.