Tissue Engineering Part A: Research Advances

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

Cellular, compositional, and mechanical gradients are found throughout biological tissues, especially in transition zones between tissue types. Yet, strategies to engineer such gradients have proven difficult due to the complex nature of these tissues. Current strategies for tissue engineering complex gradients often utilize stem cells; however, these multipotent cells require direction from environmental cues, which can be difficult to control both in vitro and in vivo. In this study, we utilize clustered regularly-interspaced short palindromic repeats (CRISPR)-guided gene modulation to direct the differentiation of multipotent adipose-derived stem cells (ASCs) to demonstrate the effectiveness of CRISPR-engineered cells in tissue engineering applications. Specifically, we screen CRISPR-interference (CRISPRi) constructs targeting the promotors of selected osteogenic inhibitors and demonstrate that ASC osteogenic differentiation and mineral deposition can be regulated with CRISPRi targeting of Noggin without the use of exogenous growth factors in tissue engineered constructs. As a proof of concept, we combine three technologies developed out of our laboratories to demonstrate the controlled deposition of these engineered cells in a gradient with CRISPR-activation multiplex-engineered aggrecan/collagen type-II-chondrogenic ASCs on a high density anisotropic type I collagen construct to create a cell and tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the enthesis. Our results display the promise of CRISPR-engineered ASCs to produce tissue gradients, similar to what is observed in native tissue.

Cell aggregates are widely used to study heterotypic cellular interactions during the development of vascularization in vitro. In this study, we examined heterotypic cellular spheroids made of adipose-derived stem cells and CD34⁺/CD31⁻ endothelial progenitor cells induced by the transfection of miR-148b mimic for de novo induction of osteogenic differentiation and miR-210 mimic for de novo induction of endotheliogenesis, respectively. The effect of the microRNA (miRs) mimic treatment group and induction time on codifferentiation was assessed in spheroids formed of transfected cells over the course of a 4-week culture. Based on gene and protein markers of osteogenic and endotheliogenic differentiation, as well as mineralization assays, our results showed that miRs directed cell differentiation and that progenitor maturity influenced the development of heterotypic cellular regions in aggregates. Overall, the success of coculture to create a prevascularized bone model is dependent on a number of factors, particularly the induction time of differentiation before combining the multiple cell types in aggregates. The approach that has been proposed could be valuable in creating vascularized bone tissue by employing spheroids as the building blocks of more complex issues through the use of cutting-edge methods such as 3D bioprinting.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through ¹³C-metabolic flux analysis (¹³C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-¹³C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out ¹³C-MFA under varying levels of nutrient availability. ¹³C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic–anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect (“inverse Crabtree effect”). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes—a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

Ischemic stroke is a devastating medical condition with poor prognosis due to the lack of effective treatment modalities. Transplantation of human neural stem cells or primary neural cells is a promising treatment approach, but this is hindered by limited suitable cell sources and low in vitro expansion capacity. This study aimed (1) use small molecules (SM) to reprogram gingival mesenchymal stem cells (GMSCs) commitment to the neural lineage cells in vitro, and (2) use hyaluronic acid (HA) hydrogel scaffolds seeded with GMSCs-derived neural lineage cells to treat ischemic stroke in vivo. Neural induction was carried out with a SM cocktail-based one-step culture protocol over a period of 24 h. The induced cells were analyzed for expression of neural markers with immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The Sprague-Dawley (SD) rats (n = 100) were subjected to the middle cerebral artery occlusion (MCAO) reperfusion ischemic stroke model. Then, after 8 days post-MCAO, the modeled rats were randomly assigned to six study groups (n = 12 per group): (1) GMSCs, (2) GMSCs-derived neural lineage cells, (3) HA and GMSCs-derived neural lineage cells, (4) HA, (5) PBS, and (6) sham transplantation control, and received their respective transplantation. Evaluation of post-stroke recovery were performed by behavioral tests and histological assessments. The morphologically altered nature of neural lineages has been observed of the GMSCs treated with SMs compared to the untreated controls. As shown by the qRT-PCR and immunocytochemistry, SMs further significantly enhanced the expression level of neural markers of GMSCs as compared with the untreated controls (all p < 0.05). Intracerebral injection of self-assembling HA hydrogel carrying GMSCs-derived neural lineage cells promoted the recovery of neural function and reduced ischemic damage in rats with ischemic stroke, as demonstrated by histological examination and behavioral assessments (all p < 0.05). In conclusion, the SM cocktail significantly enhanced the differentiation of GMSCs into neural lineage cells. The HA hydrogel was found to facilitate the proliferation and differentiation of GMSCs-derived neural lineage cells. Furthermore, HA hydrogel seeded with GMSCs-derived neural lineage cells could promote tissue repair and functional recovery in rats with ischemic stroke and may be a promising alternative treatment modality for stroke.

Spinal cord injury (SCI), caused by significant physical trauma, as well as other pathological conditions, results in electrical signaling disruption and loss of bodily functional control below the injury site. Conductive biomaterials have been considered a promising approach for treating SCI, owing to their ability to restore electrical connections between intact spinal cord portions across the injury site. In this study, we evaluated the ability of a conductive hydrogel, poly-3-amino-4-methoxybenzoic acid-gelatin (PAMB-G), to restore electrical signaling and improve neuronal regeneration in a rat SCI model generated using the compression clip method. Gelatin or PAMB-G was injected at the SCI site, yielding three groups: Control (saline), Gelatin, and PAMB-G. During the 8-week study, PAMB-G, compared to Control, had significantly lower proinflammatory factor expression, such as for tumor necrosis factor -α (0.388 ± 0.276 for PAMB-G vs. 1.027 ± 0.431 for Control) and monocyte chemoattractant protein (MCP)-1 (0.443 ± 0.201 for PAMB-G vs. 1.662 ± 0.912 for Control). In addition, PAMB-G had lower astrocyte and microglia numbers (35.75 ± 4.349 and 40.75 ± 7.890, respectively) compared to Control (50.75 ± 6.5 and 64.75 ± 10.72) and Gelatin (48.75 ± 4.787 and 71.75 ± 7.411). PAMB-G-treated rats also had significantly greater preservation and regeneration of remaining intact neuronal tissue (0.523 ± 0.059% mean white matter in PAMB-G vs 0.377 ± 0.044% in Control and 0.385 ± 0.051% in Gelatin) caused by reduced apoptosis and increased neuronal growth-associated gene expression. All these processes stemmed from PAMB-G facilitating increased electrical signaling conduction, leading to locomotive functional improvements, in the form of increased Basso–Beattie–Bresnahan scores and steeper angles in the slope test (76.667 ± 5.164 for PAMB-G, vs. 59.167 ± 4.916 for Control and 58.333 ± 4.082 for Gelatin), as well as reduced gastrocnemius muscle atrophy (0.345 ± 0.085 for PAMB-G, vs. 0.244 ± 0.021 for Control and 0.210 ± 0.058 for Gelatin). In conclusion, PAMB-G injection post-SCI resulted in improved electrical signaling conduction, which contributed to lowered inflammation and apoptosis, increased neuronal growth, and greater bodily functional control, suggesting its potential as a viable treatment for SCI.