Tissue Engineering Part A: Research Advances

In this study, we explored whether embryonic stem cell-derived mesenchymal stem cell (ES-MSC) cellular spheroids in combination with a closed chamber system could be used to create scaffold-free cartilage and endochondral graft tissues. ES-MSC cellular spheroids were cultured in chondrogenic medium for 3–4 days and seeded into a customizable Net Mold chamber system (NCS) and cultured in chondrogenic medium for an additional 18 days to fuse and form a single tissue construct. To assess potential for cartilage repair, cellular spheroids were matured in the NCS for only 7 days before implantation into ex vivo human cartilage defects. To engineer osteochondral tissues, cellular spheroids were initially cultured in chondrogenic medium for 14 days, seeded into one well of the NSC, and cultured together in osteogenic medium for 21 days. For the chondrogenic phase, cellular spheroids were initially cultured in chondrogenic medium for 14 days before seeding in an NCS chamber, adjacent to the osteogenic spheroids. The combined osteogenic and chondrogenic constructs were cultured in serum-free medium for an additional 3 weeks. Cellular spheroids cultured in the NCS developed into neocartilage tissues expressing cartilage-associated genes (COL2A1, ACAN, and COMP) and stained positive for cartilage matrix molecules (glycosaminoglycan and collagen type II). The cartilage-like constructs that were implanted into cartilage defects created in ex vivo osteoarthritic (OA) tissue resulted in repair tissue with an elastic modulus of 46 ± 6 kPa that was histologically integrated with the explant tissues. Spheroids cultured in osteogenic medium produced tissues that were positive for von Kossa stain and for osteopontin immunostaining. Pre-differentiation in chondrogenic and osteogenic medium before placing in the NCS resulted in fused cartilage and bone-like constructs with regional production of chondrogenic and mineralized matrix (Alizarin Red S, von Kossa, and osteopontin positive). Spheroids in stacked NCS chambers produced osteochondral neotissues up to 2 mm in thickness. Our results indicate the potential for cellular spheroids, from a clinically relevant ES-MSC source, to generate scaffold-free chondrogenic or osteochondrogenic graft tissues.

The aim of this study was to grow axially vascularized soft tissue flaps in sheep using the arteriovenous loop (AVL) technique to be transplanted for defect reconstruction. This technique may be a promising alternative to conventional free flaps to further reduce flap donor site morbidity. In this pilot study, AVLs (n = 12) were created in the groins of six sheep, placed into an isolation chamber, and embedded in Matriderm®. Tissue volume, vascularization, and cell proliferation were assessed on postoperative day (POD) 28 using immunohistochemical staining and microcomputed tomography (µCT). Four AVL free flaps were microsurgically anastomosed to the neck vessels in a standardized defect sheep model on POD 28. Defect closure and intrinsically vascularized scaffold-based bioengineered flaps (IVSBs) flap perfusion were studied by angiography and histology 10 days after transplantation. One IVSB flap was lost due to chamber infection. At POD 28, the remaining 11 IVSB flaps had filled the isolation chamber. Histological examination and µCT analysis of seven IVSB flaps verified homogeneous microvascular networks within the flaps. The mean number of microvessels, vessel volume, and the percentage of proliferating cells increased significantly over time. In the defect model, all four transplanted flaps showed macroscopically, angiographically, and histologically stable defect closure 10 days after transplantation, with homogeneous vascular integration into the surrounding tissue. This pilot study demonstrates that in a large animal model complex, defects can be reconstructed using free IVSB flaps with a clinically relevant tissue volume. These data provide the preclinical proof prior to human application.

Cell and tissue engineering therapies provide promise for regenerating damaged intervertebral disc (IVD) tissue and resolving the low back pain that often accompanies it. However, these treatments remain experimental and unavailable for patients. Furthermore, the large body of work characterizing and utilizing mesenchymal stromal cells (MSCs) for these applications has, unfortunately, not resulted in any FDA-approved spinal therapies. Herein, we characterized DiscGenics’s human cadaver-derived discogenic nucleus pulposus (NP) progenitor cells and, for the first time, their discogenic annulus fibrosus (AF) progenitor cells. We then used these discogenic NP and AF cells to create biomimetic human-sized total tissue-engineered IVD replacements, also known as endplate-modified angle ply structures (eDAPS), and compared these with eDAPS formulated with goat or human MSCs. Prior to eDAPS fabrication, discogenic cells were expanded using either two-dimensional attachment culture or three-dimensional suspension culture. Currently, no data exist as to how these discogenic progenitor cells deposit extracellular matrix in a 3D culture environment, nor do data exist characterizing whether the unique expansion environment influences subsequent discogenic cell behavior. Our data support that NP and AF discogenic cells occupy unique niches and serve distinct functions, both in the IVD and in an in vitro 3D culture environment. As a result, discogenic cells deposited more matrix overall than did MSCs. That matrix was distinct between the NP and AF analogs of the tissue-engineered IVDs while also being more homogeneous within each region. Most importantly, unlike both MSC groups, discogenic cells deposited little to no collagen X, suggesting that discogenic eDAPS possess a more stable regional phenotype that will be less susceptible to hypertrophy and downstream calcification. Overall, DiscGenics’s discogenic NP and AF cells made compositionally and mechanically superior eDAPS when compared with both human and goat MSCs, with only minor differences between attachment- and suspension-derived discogenic cell eDAPS, supporting their use as a cell source for the creation of human-scale living whole disc replacements.

Biological materials composed of extracellular matrix (ECM) or its components have been successfully used for tissue repair and reconstruction. Preclinical studies, along with a cohort study following stage T1A esophageal adenocarcinoma (EAC) resection, have shown that ECM biomaterials can restore esophageal mucosa and submucosa without cancer recurrence. However, the molecular mechanisms underlying these effects remain largely unexplored. The present study investigates the in vitro effects of ECM degradation products from nonmalignant esophageal (eECM) and urinary bladder (ubECM) sources on EAC cell proliferation, migration, and associated signaling pathways. Both eECM and ubECM significantly inhibited OE33 cell proliferation, with eECM exhibiting a stronger effect—reducing proliferation to 25% at 24 h and 7% at 72 h compared with pepsin control (p < 0.001). A high-throughput cell surface marker screen followed by gene and protein expression analysis revealed that both ECM sources downregulated CD164 and CXCR4, reducing CXCR4 protein levels by approximately 50% (p = 0.006 for eECM, p = 0.007 for ubECM). Notably, only eECM significantly suppressed OE33 cell migration (p ≤ 0.0001) and downregulated bone morphogenetic protein 4 BMP4 expression, along with its downstream targets pSMAD1/5/8, ID2, and SNAI2, thereby reducing epithelial–mesenchymal transition. These findings support the concept that biochemical cues from nonmalignant ECM modulate neoplastic cell behavior. Given the involvement of PI3K-Akt and BMP4 signaling in EAC progression, ECM-based strategies may warrant further investigation as potential therapeutic approaches following esophageal cancer resection.

Cartilage and osteochondral disorders pose an increasing clinical, economic, and social challenge. With an aging population, there is a need to develop innovative, nonsurgical strategies for treating defects, fractures, and other osteochondral disorders. Bone implants require surgical intervention, carry a risk of complications, are expensive, and do not always provide comfort to the patient. Alternatively, stimulation of bone regeneration using synthetic peptides is a promising and less invasive option in the treatment of trauma, orthopedics, craniofacial surgery, and dentistry. In the present study, we evaluated the biological effects of two peptides: the novel peptide UG27 and CDP4 derived from the protein Cpne7 (Copine 7), on the activity of adipose tissue-derived mesenchymal stem cells. Using labeling, differentiation, and imaging methods, we demonstrated the effect of UG27 on viability, biomineralization, extracellular matrix, and calcium salt growth in osteocytes. The peptides were immunologically safe and stimulated cell migration without showing any cytotoxic effects. The peptide, UG27, has an active connection with the biomaterial and is a promising compound in bone injury therapies.

This study has significant translational potential, combining adipose-derived stem cells with an osteoinductive peptide featuring a controlled-release linker, leading to the repair of bone defects. The peptide promotes osteogenic differentiation and may aid osteocyte cultivation by stimulating a collagen- and calcium-rich extracellular matrix. This less invasive, personalized approach could accelerate healing compared to traditional bone grafts. These findings could benefit osteoporotic patients, those undergoing long-term steroid therapy, those with bone defects and fractures, and dental patients following tooth extractions.

Osteochondral explants can serve as valuable ex vivo models for investigating joint development and testing therapeutic interventions in osteoarthritis (OA). The incorporation of synovial tissue in coculture settings more closely reproduces the inflammatory milieu characteristic of OA joints; however, no report exists regarding the culture media that can support such ex vivo systems. We investigated the reactivity of osteochondral explants using two media types: Dulbecco’s modified essential medium (DMEM) and chondrogenic medium (CHONDRO). Additionally, we tested the potential therapeutic effect of serum-free conditioned media (CM) derived from allogeneic adipose-derived stem cells (ADSCs) in the context of OA. Osteochondral fragments with or without homologous synovium were cultured in DMEM and CHONDRO for up to 30 days. A subset of explants received treatment with CM. Explant reactivity was assessed by cytokine release, synovial cellularity, and osteochondral protein content using Western blot and immunohistochemistry. Explants kept in DMEM displayed diminished levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα), together with increased Collagen II (Col II) expression. Notably, consistent suppression of TNFα was observed following CM treatment. Conversely, the CHONDRO-kept samples exhibited an increased prevalence of chondrocyte clusters; heightened Perlecan presence as well as IL-1β levels in response to CM treatment and synovial tissue-dependent fluctuations in Col II levels. Remarkably, significantly increased β-galactosidase levels could be detected in osteochondral tissues treated with CM, regardless of the culture media type. In the experimental conditions created, DMEM provided a neutral milieu and was less prone to confounding experimental outcomes, rendering it suitable for evaluating potential therapies. CHONDRO apparently increased chondrocyte clusters and facilitated extracellular matrix synthesis; however, its usage requires caution due to potential interference with experimental readouts. CM could exert an antisenescence effect, an effect that warrants further investigation.