Providing a functional pericellular matrix (PCM) by fine-tuning the microenvironment of the articular chondrocytes (ACs) can greatly improve the outcomes of articular cartilage tissue engineering. While harvesting ACs with their PCM (chondrons) results in a low cell yield and a heterogeneous mixture of ACs and chondrons, microscale hydrogels could be used for mechanical tuning of the cell microenvironment. This may enable the use of stiffer bulk materials, improving the load-bearing capacity of the construct. This study investigates the effect of microenvironmental stiffness, independent of total construct stiffness, on the regenerative performance of ACs (ECM and PCM synthesis). Additionally, we explored articular cartilage-derived progenitor cells (ACPCs) as a possible alternative to ACs in the presented system. ACs were cultured in a soft or stiff bulk hydrogel (GelMA) or were encapsulated in soft microgels and seeded into the stiff GelMA. Constructs were seeded in an ex vivo porcine chondral defect model and cultured for 28 days with dynamic mechanical stimulation using a compression-sliding bioreactor. PCM and ECM quality were assessed through cell content analysis, immunofluorescent staining, histology, and measurements of GAG and collagen content. Cell encapsulation influenced ECM synthesis and PCM amount and completeness throughout the construct. Although the nonencapsulated groups showed stronger overall alcian blue staining, the encapsulated groups demonstrated more uniform matrix deposition throughout the depth of the tissue. Furthermore, ACPCs performed similarly to ACs. These findings suggest that the approach to differentially tune encapsulating and bulk hydrogel properties holds potential for future articular cartilage tissue engineering, and that ACPCs could be used as an alternative cell source.
Fibrosis and angiogenesis are key contributors to synovial inflammation in both the early and progressive stages of rheumatoid arthritis (RA) and osteoarthritis (OA), making them important therapeutic targets to mitigate joint tissue damage. In vitro drug screening, particularly for antifibrotic and antiangiogenic efficacy, is a standard method for evaluating therapeutic candidates prior to in vivo testing. Traditionally, most studies have relied on two-dimensional (2D) monolayer cell cultures, which lack physiologically relevant cell-matrix and cell-cell interactions. Substantial evidence now indicates that three-dimensional (3D) culture systems more accurately recapitulate the structural and functional complexity of native tissue environments. We employed 3D spheroid culture models of fibrosis and neovascularization to evaluate the antiangiogenic and antifibrotic effects of pirfenidone (PFD), an FDA-approved drug for idiopathic pulmonary fibrosis. Spheroid monocultures of 3T3 fibroblasts and co-cultures of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (SMCs) were encapsulated in cell-adhesive, proteolytically degradable polyethylene glycol (PEG) hydrogel scaffolds. The temporal effects of PFD dose and timing of addition in culture on fibroblast outgrowth, vascular sprouting, and viability were quantified up to 14 days. PFD treatment led to dose-dependent inhibition of both fibroblast outgrowth and vascular sprouting, depending on the initial timing of PFD addition, with cell viability maintained under all conditions. In addition, PFD reversed the onset of fibrosis and neovascularization. PFD exhibited antifibrotic activity and antiangiogenic potential in 3D cultures.
Objective:Reconstruction of critical-sized bone defects, particularly in the cranio-maxillofacial region, presents unique challenges due to the need for integration with adjacent well-vascularized tissue and the absence of significant load-bearing requirements. This study evaluated the clinical readiness of bone tissue engineering (BTE) for critically sized cranial defects using custom 3D-printed hydroxyapatite scaffolds augmented with either recombinant human bone morphogenetic protein-2 (rhBMP-2) or dipyridamole (DIPY) in a highly translational nonhuman primate model. Methods:Identical 5 × 5-cm vertex guided craniotomies were created in 12 macaques: Three cynomolgus macaques served as negative controls to validate the critical size nature of the defect, while nine rhesus macaques underwent scaffold reconstruction. Subjects were divided into three groups: uncoated scaffolds (n = 3), scaffolds augmented with rhBMP-2 (Infuse® Medtronic, n = 3), and scaffolds coated with DIPY, an adenosine A2A receptor (A2AR) indirect agonist (n = 3). Bone growth and integration were assessed over 12 months through serial CT scans, followed by ex vivo micro-CT scanning, histology, and nanoindentation testing. Results:Negative control subjects did not demonstrate new bone formation, confirming the critical defect model. Subjects treated with scaffolds through all treatment groups remained intact throughout the 12-month follow-up. The rhBMP-2-treated group exhibited bridging, ∼90% circumferentially, significantly greater than DIPY (∼9%) or the uncoated scaffold (10%) (p < 0.001). Bone volume within rhBMP-2-treated scaffolds (7621 ± 145 mm3) significantly exceeded that of DIPY (6466 ± 693 mm3, p = 0.03) and uncoated scaffold (6348 ± 663 mm3, p = 0.02) groups at 12 months. Quantitative histological micrograph analysis demonstrated that rhBMP-2 scaffolds were associated with the highest bone ingrowth (∼64%) relative to DIPY (∼39%) and uncoated scaffolds (∼27%). Nanoindentation yielded superior mechanical properties (Young's modulus and hardness) of newly generated bone with defects treated with rhBMP-2 scaffolds (p < 0.05). Conclusions:Reconstructing critically sized cranial defects with custom 3D-printed hydroxyapatite scaffolds was successful and yielded favorable results in this model. Scaffolds augmented with rhBMP-2 demonstrated superior bone ingrowth, integration, and mechanical properties, highlighting their potential as a viable alternative to autografts and allograft materials for cranioplasty.
Chronic respiratory diseases are a major global health concern. Lung epithelial dysfunction is a common underlying feature of many such conditions; hence, reconstructing the diseased epithelium with functional epithelial cells is a promising therapeutic approach. There are various endogenous stem cell and progenitor populations in the lung epithelium that can be utilized for transplantation. Additionally, pluripotent stem cells (PSCs) have emerged as a valuable source for generating therapeutic cells due to their capacity for indefinite self-renewal and the availability of directed differentiation protocols to transform them into lung progenitors or mature lung epithelial cells. This review discusses the endogenous stem cell and progenitor populations of the lung epithelium, recent advances in developing directed differentiation protocols to generate these cells, and the application of both endogenous and PSC-derived lung epithelial cells for disease modeling in vitro and as cell therapies in vivo. It provides valuable insights into the current progress of regenerative medicine within the respiratory field and highlights areas that require further research.
