Non-neotissue constituents as underestimated confounders in the assessment of tissue engineered constructs by near-infrared spectroscopy.

Non-destructive assessments are required for the quality control of tissue-engineered constructs and the optimization of the tissue culture process. Near-infrared (NIR) spectroscopy coupled with machine learning (ML) provides a promising approach for such assessment. However, due to its nonspecific nature, each spectrum incorporates information on both neotissue and non-neotissue constituents of the construct; the effect of these constituents on the NIR-based assessments of tissue-engineered constructs has been overlooked in previous studies. This study investigates the effect of scaffolds, growth factors, and buffers on NIR-based assessments of tissue-engineered constructs. To determine if these non-neotissue constituents have a measurable effect on the NIR spectra of the constructs that can introduce bias in their assessment, nine ML algorithms were evaluated in classifying the NIR spectra of engineered cartilage according to the scaffold used to prepare the constructs, the growth factors added to the culture media, and the buffers used for storing the constructs. The effect of controlling for these constituents was also evaluated using controlled and uncontrolled NIR-based ML models for predicting tissue maturity as an example of neotissue-related properties of interest. Samples used in this study were prepared using norbornene-modified hyaluronic acid scaffolds with or without the conjugation of an N-cadherin mimetic peptide. Selected samples were supplemented with transforming growth factor-beta1 or bone morphogenetic protein-9 growth factor. Some samples were frozen in cell lysis buffer, while the remaining samples were frozen in PBS until required for NIR analysis. The ML models for classifying the spectra of the constructs according to the four constituents exhibited high to fair performances, with F1 scores ranging from 0.9 to 0.52. Moreover, controlling for the four constituents significantly improved the performance of the models for predicting tissue maturity, with improvement in F1 scores ranging from 0.09 to 0.77. In conclusion, non-neotissue constituents have measurable effects on the NIR spectra of tissue-engineered constructs that can be detected by ML algorithms and introduce bias in the assessment of the constructs by NIR spectroscopy. Therefore, controlling for these constituents is necessary for reliable NIR-based assessments of tissue-engineered constructs.

Modulating design parameters to drive cell invasion into hydrogels for osteochondral tissue formation.

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p â€‹= â€‹0.390) and led to a higher migration score ex vivo (p â€‹< â€‹0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p â€‹= â€‹0.031) and ex vivo (p â€‹< â€‹0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p â€‹< â€‹0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used.The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application.

Differential Production of Cartilage ECM in 3D Agarose Constructs by Equine Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells.

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.

One-Step Photoactivation of a Dual-Functionalized Bioink as Cell Carrier and Cartilage-Binding Glue for Chondral Regeneration.

Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration-permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible-light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual-functionalized tyramine and methacryloyl gelatin (GelMA-Tyr) is synthesized. Photo-crosslinking of both groups is triggered in a single photoexposure by cell-compatible visible light in presence of tris(2,2'-bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo-cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion-printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365-400 nm range), commonly used in biofabrication. Tyramine-binding to proteins in native cartilage leads to a 15-fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible-light reactive GelMA-Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.

Mimicking the Articular Joint with In Vitro Models.

Treating joint diseases remains a significant clinical challenge. Conventional in vitro cultures and animal models have been helpful, but suffer from limited predictive power for the human response. Advanced models are therefore required to mimic the complex biological interactions within the human joint. However, the intricate structure of the joint microenvironment and the complex nature of joint diseases have challenged the development of in vitro models that can faithfully mimic the in vivo physiological and pathological environments. In this review, we discuss the current in vitro models of the joint and the progress achieved in the development of novel and potentially more predictive models, and highlight the application of new technologies to accurately emulate the articular joint.