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Objective: The consequences of racism and racial stress on the academic and social well-being of adolescents are profound and well-documented. However, our understanding of how adolescents navigate racial stress and develop the agency to address discriminatory encounters, particularly in settings where educators struggle to intervene with microaggressions, remains limited. Research into the development of racial coping self-efficacy (RCSE) and coping skills has shown promise in enhancing the overall well-being of youth. In this study, we employ racial encounter coping appraisal and socialization theory (RECAST) to investigate the relationships among key school-based factors that influence how students cope with racial challenges. Specifically, we examine the role of RCSE, agency, stress, vigilance, and sense of belonging in the school environment for students., Method: Participants included 645 high school students from a diverse public school district who completed an online survey during a single class period. Analyses examined the mediating and moderating effects of RCSE, racial coping stress (RCS), and racial vigilance on agency., Results: RCSE reduced the negative influence of RCS on student racial agency in resolving racial conflicts with teachers and peers, particularly for Latinx students., Conclusions: Implications for advancing RECAST-based interventions to help adolescents develop the agency to problem solve traumatic racial events in schools are discussed. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

The tailorable properties of synthetic polyethylene glycol (PEG) hydrogels make them an attractive substrate for human organoid assembly. Here, we formed human neural organoids from iPSC-derived progenitor cells in two distinct formats: (i) cells seeded on a Matrigel surface; and (ii) cells seeded on a synthetic PEG hydrogel surface. Tissue assembly on synthetic PEG hydrogels resulted in three dimensional (3D) planar neural organoids with greater neuronal diversity, greater expression of neurovascular and neuroinflammatory genes, and reduced variability when compared with tissues assembled upon Matrigel. Further, our 3D human tissue assembly approach occurred in an open cell culture format and created a tissue that was sufficiently translucent to allow for continuous imaging. Planar neural organoids formed on PEG hydrogels also showed higher expression of neural, vascular, and neuroinflammatory genes when compared to traditional brain organoids grown in Matrigel suspensions. Further, planar neural organoids contained functional microglia that responded to pro-inflammatory stimuli, and were responsive to anti-inflammatory drugs. These results demonstrate that the PEG hydrogel neural organoids can be used as a physiologically relevant in vitro model of neuro-inflammation., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: W.L.M. and C.S.L. are co-founders and shareholders in Stem Pharm, Inc., which is focused on commercial applications of neural organoids. C.S.L., P.F.F., and W.D.R. are employees of Stem Pharm, Inc., (© The Author(s) 2024.)

Pericytes play a critical role in promoting, regulating, and maintaining numerous vascular functions. Their dysfunction is a major contributor to the progression of vascular and neurodegenerative diseases, making them an ideal candidate for large-scale production for disease modeling and regenerative cell therapy. This protocol describes the rapid and robust differentiation of pericytes from human induced pluripotent stem cells (hiPSCs) while simultaneously generating a population of hiPSC-derived endothelial progenitor cells. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2017)., Competing Interests: W.L.M. is co-founder and chief scientific officer at Stem Pharm, Inc. and Dianomi Therapeutics, Inc. Patents are pending for intellectual property described in this report., (© 2020 The Author(s).)

Matrigel, a basement-membrane matrix extracted from Engelbreth-Holm-Swarm mouse sarcomas, has been used for more than four decades for a myriad of cell culture applications. However, Matrigel is limited in its applicability to cellular biology, therapeutic cell manufacturing and drug discovery owing to its complex, ill-defined and variable composition. Variations in the mechanical and biochemical properties within a single batch of Matrigel - and between batches - have led to uncertainty in cell culture experiments and a lack of reproducibility. Moreover, Matrigel is not conducive to physical or biochemical manipulation, making it difficult to fine-tune the matrix to promote intended cell behaviours and achieve specific biological outcomes. Recent advances in synthetic scaffolds have led to the development of xenogenic-free, chemically defined, highly tunable and reproducible alternatives. In this Review, we assess the applications of Matrigel in cell culture, regenerative medicine and organoid assembly, detailing the limitations of Matrigel and highlighting synthetic scaffold alternatives that have shown equivalent or superior results. Additionally, we discuss the hurdles that are limiting a full transition from Matrigel to synthetic scaffolds and provide a brief perspective on the future directions of synthetic scaffolds for cell culture applications.

Human induced pluripotent stem cells (iPSCs) have emerged as a promising alternative to bone-marrow derived mesenchymal stem/stromal cells for cartilage tissue engineering. However, the effect of biochemical and mechanical cues on iPSC chondrogenesis remains understudied. This study evaluated chondrogenesis of induced pluripotent mesenchymal progenitor cells (iPS-MPs) encapsulated in a cartilage-mimetic hydrogel under different culture conditions: free swelling versus dynamic compressive loading and different growth factors (TGFβ3 and/or BMP2). Human iPSCs were differentiated into iPS-MPs and chondrogenesis was evaluated by gene expression (qPCR) and protein expression (immunohistochemistry) after three weeks. In pellet culture, both TGFβ3 and BMP2 were required to promote chondrogenesis. However, the hydrogel in growth factor-free conditions promoted chondrogenesis, but rapidly progressed to hypertrophy. Dynamic loading in growth factor-free conditions supported chondrogenesis, but delayed the transition to hypertrophy. Findings were similar with TGFβ3, BMP2, and TGFβ3 + BMP2. Dynamic loading with TGFβ3, regardless of BMP2, was the only condition that promoted a stable chondrogenic phenotype (aggrecan + collagen II) accompanied by collagen X down-regulation. Positive TGFβRI expression with load-enhanced Smad2/3 signaling and low SMAD1/5/8 signaling was observed. In summary, this study reports a promising cartilage-mimetic hydrogel for iPS-MPs that when combined with appropriate biochemical and mechanical cues induces a stable chondrogenic phenotype.

Focal defects in articular cartilage are unable to self-repair and, if left untreated, are a leading risk factor for osteoarthritis. This study examined cartilage degeneration surrounding a defect and then assessed whether infilling the defect prevents degeneration. We created a focal chondral defect in porcine osteochondral explants and cultured them ex vivo with and without dynamic compressive loading to decouple the role of loading. When compared to a defect in a porcine knee four weeks post-injury, this model captured loss in sulfated glycosaminoglycans (sGAGs) along the defect's edge that was observed in vivo, but this loss was not load dependent. Loading, however, reduced the indentation modulus of the surrounding cartilage. After infilling with in situ polymerized hydrogels that were soft (100 kPa) or stiff (1 MPa) and which produced swelling pressures of 13 and 310 kPa, respectively, sGAG loss was reduced. This reduction correlated with increased hydrogel stiffness and swelling pressure, but was not affected by loading. This ex vivo model recapitulates sGAG loss surrounding a defect and, when infilled with a mechanically supportive hydrogel, degeneration is minimized., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) are promising for cartilage regeneration, but readily undergo terminal differentiation. The aim of this study was two-fold: a) investigate physiochemical cues from a cartilage-mimetic hydrogel under dynamic compressive loading on MSC chondrogenesis and hypertrophy and b) identify whether Smad signaling and p38 MAPK signaling mediate hypertrophy during MSC chondrogenesis. Human MSCs were encapsulated in photoclickable poly(ethylene glycol) hydrogels containing chondroitin sulfate and RGD, cultured under dynamic compressive loading or free swelling for three weeks, and evaluated by qPCR and immunohistochemistry. Loading inhibited hypertrophy in the cartilage-mimetic hydrogel indicated by a reduction in pSmad 1/5/8, Runx2, and collagen X proteins, while maintaining chondrogenesis by pSmad 2/3 and collagen II proteins. Inhibiting pSmad 1/5/8 under free swelling culture significantly reduced collagen X protein, similar to the loading condition. Chondroitin sulfate was necessary for load-inhibited hypertrophy and correlated with enhanced S100A4 expression, which is downstream of the osmotic responsive transcription factor NFAT5. Inhibiting p38 MAPK under loading reduced S100A4 expression, and upregulated Runx2 and collagen X protein. Findings from this study indicate that chondroitin sulfate with dynamic loading create physiochemical cues that support MSC chondrogenesis and attenuate hypertrophy through Smad 1/5/8 inhibition and p38 MAPK upregulation., (Copyright © 2018. Published by Elsevier Ltd.)

Background: In this study, we investigate the in vitro and in vivo chondrogenic capacity of a novel photopolymerizable cartilage mimetic hydrogel, enhanced with extracellular matrix analogs, for cartilage regeneration., Purpose: To (1) determine whether mesenchymal stem cells (MSCs) embedded in a novel cartilage mimetic hydrogel support in vitro chondrogenesis, (2) demonstrate that the proposed hydrogel can be delivered in situ in a critical chondral defect in a rabbit model, and (3) determine whether the hydrogel with or without MSCs supports in vivo chondrogenesis in a critical chondral defect., Study Design: Controlled laboratory study., Methods: Rabbit bone marrow-derived MSCs were isolated, expanded, encapsulated in the hydrogel, and cultured in chondrogenic differentiation medium for 9 weeks. Compressive modulus was evaluated at day 1 and at weeks 3, 6, and 9. Chondrogenic differentiation was investigated via quantitative polymerase reaction, safranin-O staining, and immunofluorescence. In vivo, a 3 mm-wide × 2-mm-deep chondral defect was created bilaterally on the knee trochlea of 10 rabbits. Each animal had 1 defect randomly assigned to be treated with hydrogel with or without MSCs, and the contralateral knee was left untreated. Hence, each rabbit served as its own matched control. Three groups were established: group A, hydrogel (n = 5); group B, hydrogel with MSCs (n = 5); and group C, control (n = 10). Repair tissue was evaluated at 6 months after intervention., Results: In vitro, chondrogenesis and the degradable behavior of the hydrogel by MSCs were confirmed. In vivo, the hydrogel could be delivered intraoperatively in a sterile manner. Overall, the hydrogel group had the highest scores on the modified O'Driscoll scoring system (group A, 17.4 ± 4.7; group B, 13 ± 3; group C, 16.7 ± 2.9) ( P = .11) and showed higher safranin-O staining (group A, 49.4% ± 20%; group B, 25.8% ± 16.4%; group C, 36.9% ± 25.2%) ( P = .27), although significance was not detected for either parameter., Conclusion: This study provides the first evidence of the ability to photopolymerize this novel hydrogel in situ and assess its ability to provide chondrogenic cues for cartilage repair in a small animal model. In vitro chondrogenesis was evident when MSCs were encapsulated in the hydrogel., Clinical Relevance: Cartilage mimetic hydrogel may offer a tissue engineering approach for the treatment of osteochondral lesions.

Cartilage tissue engineering strategies that use in situ forming degradable hydrogels for mesenchymal stem cell (MSC) delivery are promising for treating chondral defects. Hydrogels that recapitulate aspects of the native tissue have the potential to encourage chondrogenesis, permit cellular mediated degradation, and facilitate tissue growth. This study investigated photoclickable poly(ethylene glycol) hydrogels, which were tailored to mimic the cartilage microenvironment by incorporating extracellular matrix analogs, chondroitin sulfate and RGD, and crosslinks sensitive to matrix metalloproteinase 7 (MMP7). Human MSCs were encapsulated in the hydrogel, cultured up to nine weeks, and assessed by mRNA expression, protein production and biochemical analysis. Chondrogenic genes, SOX9, ACAN, and COL2A1, significantly increased with culture time, and the ratios of COL2A1:COL10A1 and SOX9:RUNX2 reached values of ∼20-100 by week 6. The encapsulated MSCs degraded the hydrogel, which was nearly undetectable by week 9. There was substantial deposition of aggrecan and collagen II, which correlated with degradation of the hydrogel. Minimal collagen X was detectable, but collagen I was prevalent. After week 1, extracellular matrix elaboration was accompanied by a ∼twofold increase in compressive modulus with culture time. The MMP7-sensitive cartilage mimetic hydrogel supported MSC chondrogenesis and promoted macroscopic neocartilaginous matrix elaboration representative of fibrocartilage. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2344-2355, 2018., (© 2018 Wiley Periodicals, Inc.)

Damage to articular cartilage can over time cause degeneration to the tissue surrounding the injury. To address this problem, scaffolds that prevent degeneration and promote neotissue growth are needed. A new hybrid scaffold that combines a stereolithography-based 3D printed support structure with an injectable and photopolymerizable hydrogel for delivering cells to treat focal chondral defects is introduced. In this proof of concept study, the ability to a) infill the support structure with an injectable hydrogel precursor solution, b) incorporate cartilage cells during infilling using a degradable hydrogel that promotes neotissue deposition, and c) minimize damage to the surrounding cartilage when the hybrid scaffold is placed in situ in a focal chondral defect in an osteochondral plug that is cultured under mechanical loading is demonstrated. With the ability to independently control the properties of the structure and the injectable hydrogel, this hybrid scaffold approach holds promise for treating chondral defects., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Focal chondral lesions and early osteoarthritis (OA) are responsible for progressive joint pain and disability in millions of people worldwide, yet there is currently no surgical joint preservation treatment available to fully restore the long term functionality of cartilage. Limitations of current treatments for cartilage defects have prompted the field of cartilage tissue engineering, which seeks to integrate engineering and biological principles to promote the growth of new cartilage to replace damaged tissue. Toward improving cartilage repair, hydrogel design has advanced in recent years to improve their utility. Injectable hydrogels have emerged as a promising scaffold due to their wide range of properties, the ability to encapsulate cells within the material, and their ability to provide cues for cell differentiation. Some of these advances include the development of improved control over in situ gelation (e.g., light), new techniques to process hydrogels (e.g., multi-layers), and better incorporation of biological signals (e.g., immobilization, controlled release, and tethering). This review summarises the innovative approaches to engineer injectable hydrogels toward cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:64-75, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Current hydrogels used for tissue engineering are limited to a single range of mechanical properties within the replicated tissue construct. We show that repeated in-swelling by a single hydrogel pre-cursor solution into an existing polymerized hydrogel followed by photo-exposure increases hydrogel mechanical properties. The process is demonstrated with a photo-clickable thiol-ene hydrogel using a biocompatible precursor solution of poly(ethylene glycol) dithiol and 8-arm poly(ethylene glycol) functionalized with norbornene. The polymer fraction in the precursor solution was varied by 5, 10, and 20 percent by weight and an off-stoichiometric ratio of thiol : ene was used, leaving free enes available for subsequent reaction. Multiple swelling and exposure cycles for the same precursor solution were performed. The compressive modulus increased by a factor between three and ten (formulation dependent), while volume swelling ratio decreased by a factor of two, consistent with increased crosslink density. The modified hydrogels also demonstrate increased toughness by fracturing at compressive forces five times greater than the initial hydrogel. We attribute the increased toughness to subsequent increases in crosslink density created by the repeated photopolymerization of in-swollen macromer. This technique demonstrates the ability to significantly modify hydrogel network properties by exploiting swelling and polymerization processes that can be applied to traditional three-dimensional printing systems to spatially control local mechanical properties.

Three dimensional hydrogels are a promising vehicle for delivery of adult human bone-marrow derived mesenchymal stem cells (hMSCs) for cartilage tissue engineering. One of the challenges with using this cell type is the default pathway is terminal differentiation, a hypertrophic phenotype and precursor to endochondral ossification. We hypothesized that a synthetic hydrogel consisting of extracellular matrix (ECM) analogs derived from cartilage when combined with dynamic loading provides physiochemical cues for achieving a stable chondrogenic phenotype. Hydrogels were formed from crosslinked poly(ethylyene glycol) as the base chemistry and to which (meth)acrylate functionalized ECM analogs of RGD (cell adhesion peptide) and chondroitin sulfate (ChS, a negatively charged glycosaminoglycan) were introduced. Bone-marrow derived hMSCs from three donors were encapsulated in the hydrogels and cultured under free swelling conditions or under dynamic com pressive loading with 2.5 ng/ml TGF-β3. hMSC differentiation was assessed by quantitative PCR and immunohistochemistry. Nine hydrogel formulations were initially screened containing 0, 0.1 or 1mM RGD and 0, 1 or 2wt% ChS. After 21 days, the 1% ChS and 0.1 mM RGD hydrogel had the highest collagen II gene expression, but this was accompanied by high collagen X gene expression. At the protein level, collagen II was detected in all formulations with ECM analogs, but minimally detectable in the hydrogel without ECM analogs. Collagen X protein was present in all formulations. The 0.1 mM RGD and 1% ChS formulation was selected and subjected to five loading regimes: no loading, 5% strain 0.3Hz (1.5%/s), 10% strain 0.3 Hz (3%/s), 5% strain 1 Hz (5%/s), and 10% strain 1Hz (10%/s). After 21 days, ~70-90% of cells stained positive for collagen II protein regardless of the culture condition. On the contrary, only ~20-30% of cells stained positive for collagen X protein under 3 and 5%/s loading conditions, which was accompanied by minimal staining for RunX2. The other culture conditions had more cells staining positive for collagen X (40-60%) and was accompanied by positive staining for RunX2. In summary, a cartilage-like biomimetic hydrogel supports chondrogenesis of hMSCs, but dynamic loading only under select strain rates is able to inhibit hypertrophy.

A bioinspired multi-layer hydrogel was developed for the encapsulation of human mesenchymal stem cells (hMSCs) as a platform for osteochondral tissue engineering. The spatial presentation of biochemical cues, via incorporation of extracellular matrix analogs, and mechanical cues, via both hydrogel crosslink density and externally applied mechanical loads, were characterized in each layer. A simple sequential photopolymerization method was employed to form stable poly(ethylene glycol)-based hydrogels with a soft cartilage-like layer of chondroitin sulfate and low RGD concentrations, a stiff bone-like layer with high RGD concentrations, and an intermediate interfacial layer. Under a compressive load, the variation in hydrogel stiffness within each layer produced high strains in the soft cartilage-like layer, low strains in the stiff bone-like layer, and moderate strains in the interfacial layer. When hMSC-laden hydrogels were cultured statically in osteochondral differentiation media, the local biochemical and matrix stiffness cues were not sufficient to spatially guide hMSC differentiation after 21 days. However dynamic mechanical stimulation led to differentially high expression of collagens with collagen II in the cartilage-like layer, collagen X in the interfacial layer and collagen I in the bone-like layer and mineral deposits localized to the bone layer. Overall, these findings point to external mechanical stimulation as a potent regulator of hMSC differentiation toward osteochondral cellular phenotypes., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)