A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis.

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

Comparison of the Translational Potential of Human Mesenchymal Progenitor Cells from Different Bone Entities for Autologous 3D Bioprinted Bone Grafts.

Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.

Infrapatellar Fat Pad Modulates Osteoarthritis-Associated Cytokine and MMP Expression in Human Articular Chondrocytes.

Osteoarthritis (OA) most frequently affects the knee joint and is associated with an elevated expression of cytokines and extracellular cartilage matrix (ECM), degrading enzymes such as matrix metalloproteinases (MMPs). Differences in gene expression of the intra-articularly located infrapatellar fat pad (IPFP) and other fatty tissue suggest its autonomous function, yet its role in OA pathogenesis remains unknown. Human IPFPs and articular cartilage were collected from OA patients undergoing total knee arthroplasty, and biopsies from the IPFP of healthy patients harvested during knee arthroscopy served as controls (CO). Isolated chondrocytes were co-cultured with either osteoarthritic (OA) or CO-IPFPs in a transwell system. Chondrocyte expression of MMP1, -3, -13, type 1 and 2 collagens, interleukin IL1ß, IL6, IL10, and tumor necrosis factor TNFα was analyzed by RTD-PCR at day 0 and day 2, and TNFα secretion was analyzed by ELISA. The cytokine release in IPFPs was assessed by an array. Results: Both IPFPs (CO, OA) significantly reduced the expression of type 2 collagen and TNFα in chondrocytes. On the other hand, only CO-IPFP suppressed the expression of type 1 collagen and significantly induced the MMP13 expression. On the contrary, IL1ß and IL6 were significantly induced when exposed to OA-IPFP. Conclusions: The partial loss of the suppressive effect on type 1 collagen gene expression found for OA-IPFP shows the pathological remodeling and dedifferentiation potential of the OA-IPFP on the chondrocytes. However, the significant suppression of TNFα implies that the OA- and CO-IPFP could also exhibit a protective role in the knee joint, preventing the progress of inflammation.

Therapeutic Approaches Targeting Vascular Repair After Experimental Spinal Cord Injury: A Systematic Review of the Literature.

Traumatic spinal cord injury (SCI) disrupts the spinal cord vasculature resulting in ischemia, amplification of the secondary injury cascade and exacerbation of neural tissue loss. Restoring functional integrity of the microvasculature to prevent neural loss and to promote neural repair is an important challenge and opportunity in SCI research. Herein, we summarize the course of vascular injury and repair following SCI and give a comprehensive overview of current experimental therapeutic approaches targeting spinal cord microvasculature to diminish ischemia and thereby facilitate neural repair and regeneration. A systematic review of the published literature on therapeutic approaches to promote vascular repair after experimental SCI was performed using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) standards. The MEDLINE databases PubMed, Embase, and OVID MEDLINE were searched using the keywords "spinal cord injury," "angiogenesis," "angiogenesis inducing agents," "tissue engineering," and "rodent subjects." A total of 111 studies were identified through the search. Five main therapeutic approaches to diminish hypoxia-ischemia and promote vascular repair were identified as (1) the application of angiogenic factors, (2) genetic engineering, (3) physical stimulation, (4) cell transplantation, and (5) biomaterials carrying various factor delivery. There are different therapeutic approaches with the potential to diminish hypoxia-ischemia and promote vascular repair after experimental SCI. Of note, combinatorial approaches using implanted biomaterials and angiogenic factor delivery appear promising for clinical translation.

Blends of gelatin and hyaluronic acid stratified by stereolithographic bioprinting approximate cartilaginous matrix gradients.

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.

CCL25 chemokine-guided stem cell attraction: an assessment of possible benefits and risks.

Due to its chemoattraction potential on mesenchymal stromal cells of the CCL25/CCR9 axis, local application of CCL25 to severely damaged tissues may be a promising approach for regenerative therapies. Analysis of the given data revealed that CCL25/CCR9 signaling has a crucial role in regulation of an adult immune homeostasis. CCR9 expression variations resulted in dysfunctional immune response in colitis, rheumatoid arthritis and endometriosis. Regarding oncology, different neoplastic tissues exploit CCL25-dependent CCR9 signaling for either local proliferation or migration processes. The CCR9 pathway likely can trigger crosstalk between the Akt and NOTCH pathway and thus participate in the regulation of the neoplastic behavior. In conclusion, the designated application-tissue requires precise molecular analysis of possible CCR9 expression due to its proto-oncogenic characteristics.