Search Results (13)

Background: A trilayered collagen/collagen–magnesium–hydroxyapatite (Col/Col-Mg-HA) scaffold is used in clinical practice to treat osteochondral lesions, but the regeneration of the subchondral bone is still not satisfactory. Objective: The aim of this study was to test, in vitro, the osteoinductivity induced by the addition of bone morphogenetic protein-2 (BMP-2) or amorphous calcium phosphate granules with strontium ions (Sr-ACP), in order to improve the clinical regeneration of subchondral bone, still incomplete. Methodology: Normal human osteoblasts (NHOsts) were seeded on the scaffolds and grown for 14 days in the presence of human osteoclasts and conditioned medium of human endothelial cells. NHOst adhesion and morphology were observed with transmission electron microscopy, and metabolic activity was tested by Alamar blue assay. The expression of osteoblast- and osteoclast-typical markers was evaluated by RT-PCR on scaffolds modified by enrichment with BPM-2 or Sr-ACP, as well as on unmodified material used as a control. Results: NHOsts adhered well to all types of scaffolds, maintained their typical morphology, and secreted abundant extracellular matrix. On the modified materials, COL1A1, SPARC, SPP1, and BGLAP were more expressed than on the unmodified ones, showing the highest expression in the presence of BMP-2. On Sr-ACP-enriched scaffolds, NHOsts had a lower proliferation rate and a lower expression of RUNX2, SP7, and ALPL compared to the other materials. The modified scaffolds, particularly the one containing Sr-ACP, increased the expression of the osteoclasts’ typical markers and decreased the OPG/RANKL ratio. Both types of scaffold modification were able to increase the osteoinductivity with respect to the original scaffold used in clinical practice. BMP-2 modification seemed to be more slightly oriented to sustain NHOst activity, and Sr-ACP seemed to be more slightly oriented to sustain the osteoclast activity. These could provide a concerted action toward better regeneration of the entire osteochondral unit. Full article

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. Full article

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models. Full article

Human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) are of great interest in tissue engineering. We obtained hWJ-MSCs from four patients, and then we stimulated their chondrogenic phenotype formation in vitro by adding resveratrol (during cell expansion) and a canonical Wnt pathway activator, LiCl, as well as a Rho-associated protein kinase inhibitor, Y27632 (during differentiation). The effects of the added reagents on the formation of hWJ-MSC sheets destined to repair osteochondral injuries were investigated. Three-dimensional hWJ-MSC sheets grown on P(NIPAM-co-NtBA)-based matrices were characterized in vitro and in vivo. The combination of resveratrol and LiCl showed effects on hWJ-MSC sheets similar to those of the basal chondrogenic medium. Adding Y27632 decreased both the proportion of hypertrophied cells and the expression of the hyaline cartilage markers. In vitro, DMSO was observed to impede the effects of the chondrogenic factors. The mouse knee defect model experiment revealed that hWJ-MSC sheets grown with the addition of resveratrol and Y27632 were well integrated with the surrounding tissues; however, after 3 months, the restored tissue was identical to that of the naturally healed cartilage injury. Thus, the combination of chondrogenic supplements may not always have additive effects on the progress of cell culture and could be neutralized by the microenvironment after transplantation. Full article

Cytotherapies are often necessary for the management of symptomatic large knee (osteo)-chondral defects. While autologous chondrocyte implantation (ACI) has been clinically used for 30 years, allogeneic cells (clinical-grade FE002 primary chondroprogenitors) have been investigated in translational settings (Swiss progenitor cell transplantation program). The aim of this study was to comparatively assess autologous and allogeneic approaches (quality, safety, functional attributes) to cell-based knee chondrotherapies developed for clinical use. Protocol benchmarking from a manufacturing process and control viewpoint enabled us to highlight the respective advantages and risks. Safety data (telomerase and soft agarose colony formation assays, high passage cell senescence) and risk analyses were reported for the allogeneic FE002 cellular active substance in preparation for an autologous to allogeneic clinical protocol transposition. Validation results on autologous bioengineered grafts (autologous chondrocyte-bearing Chondro-Gide scaffolds) confirmed significant chondrogenic induction (COL2 and ACAN upregulation, extracellular matrix synthesis) after 2 weeks of co-culture. Allogeneic grafts (bearing FE002 primary chondroprogenitors) displayed comparable endpoint quality and functionality attributes. Parameters of translational relevance (transport medium, finished product suturability) were validated for the allogeneic protocol. Notably, the process-based benchmarking of both approaches highlighted the key advantages of allogeneic FE002 cell-bearing grafts (reduced cellular variability, enhanced process standardization, rationalized logistical and clinical pathways). Overall, this study built on our robust knowledge and local experience with ACI (long-term safety and efficacy), setting an appropriate standard for further clinical investigations into allogeneic progenitor cell-based orthopedic protocols. Full article

Despite significant advances in biomedical research, osteochondral defects resulting from injury, an autoimmune condition, cancer, or other pathological conditions still represent a significant medical problem. Even though there are several conservative and surgical treatment approaches, in many cases, they do not bring the expected results and further permanent damage to the cartilage and bones occurs. Recently, cell-based therapies and tissue engineering have gradually become promising alternatives. They combine the use of different types of cells and biomaterials to induce regeneration processes or replace damaged osteochondral tissue. One of the main challenges of this approach before clinical translation is the large-scale in vitro expansion of cells without changing their biological properties, while the use of conditioned media which contains various bioactive molecules appears to be very important. The presented manuscript provides a review of the experiments focused on osteochondral regeneration by using conditioned media. In particular, the effect on angiogenesis, tissue healing, paracrine signaling, and enhancing the properties of advanced materials are pointed out. Full article

With the increase in population aging, the tendency of osteochondral injury will be accelerated, and repairing materials are increasingly needed for the optimization of the regenerative processes in bone and cartilage recovery. The local environment of the injury sites and the deficiency of Mg²⁺ retards the repairing period via inhibiting the progenitor osteogenesis and chondrogenesis cells’ recruitment, proliferation, and differentiation, which results in the sluggish progress in the osteochondral repairing materials design. In this article, we elucidate the Mg²⁺-concentration specified effect on the cell proliferation, osteochondral gene expression, and differentiation of modeling chondrocytes (extracted from New Zealand white rabbit) and osteoblasts (MC3T3-E1). The concentration of Mg²⁺ in the culture medium affects the proliferation, chondrogenesis, and osteogenesis: (i) Appropriate concentrations of Mg²⁺ promote the proliferation of chondrocytes (1.25–10.0 mM) and MC3T3-E1 cells (2.5–30.0 mM); (ii) the optimal concentration of Mg²⁺ that promotes the gene expression of noncalcified cartilage is 15 mM, calcified cartilage 10 mM, and subchondral bone 5 mM, respectively; (iii) overdosed Mg²⁺ leads to the inhibition of cell activity for either chondrocytes (>20 mM) or osteoblasts (>30 mM). The biomimetic elucidation for orchestrating the allocation of gradient concentration of Mg²⁺ in accordance of the physiological condition is crucial for designing the accurate microenvironment in osteochondral injury defects for optimization of bone and cartilage repairing materials in the future. Full article

The preservation conditions of fresh osteochondral allografts for clinical applications are critical due their objective: to transplant mature hyaline cartilage containing viable chondrocytes, maintaining their metabolic activity and also preserving the structural and functional characteristics of the extracellular matrix. The aim of the present study was to compare fluorescence confocal microscopy and flow cytometry techniques to evaluate the viability of the chondrocytes present in the osteochondral tissue, in order to determine their effectiveness and thus ensure reproducibility and robustness of the analysis. To this end, osteochondral allografts from human cadaveric donors were preserved at 4 °C for 3 weeks in a preservation medium supplemented with antibiotic and antifungal agents. Cell viability of chondrocytes was determined by monitoring the cartilage for 3 weeks of preservation by confocal fluorescence microscopy and flow cytometry, obtaining cell viabilities of 83.7 ± 2.6% and 55.8 ± 7.8% for week three, respectively. The confocal fluorescence microscopy approach is more advantageous and accurate, as it correlates better with actual cell viability values for monitoring osteochondral graft preservation, detecting only the cells that died a natural death associated with the preservation method. Full article

Osteoarthritis (OA) is a highly prevalent joint disease still lacking effective treatments. Its multifactorial etiology hampers the development of relevant preclinical models to evaluate innovative therapeutic solutions. In the last decade, the potential of Mesenchymal Stem Cell (MSC) secretome, or conditioned medium (CM), has emerged as an alternative to cell therapy. Here, we investigated the effects of the CM from adipose MSCs (ASCs), accounting for both soluble factors and extracellular vesicles, on human osteochondral explants. Biopsies, isolated from total knee replacement surgery, were cultured without additional treatment or with the CM from 10⁶ ASCs, both in the absence and in the presence of 10 ng/mL TNFα. Tissue viability and several OA-related hallmarks were monitored at 1, 3 and 6 days. Specimen viability was maintained over culture. After 3 days, TNFα induced the enhancement of matrix metalloproteinase activity and glycosaminoglycan release, both efficiently counteracted by CM. The screening of inflammatory lipids, proteases and cytokines outlined interesting modulations, driving the attention to new players in the OA process. Here, we confirmed the promising beneficial action of ASC secretome in the OA context and profiled several bioactive factors involved in its progression, in the perspective of accelerating an answer to its unmet clinical needs. Full article

Synovial mesenchymal stem cell (SMSC) is the promising cell source of cartilage regeneration but has several issues to overcome such as limited cell proliferation and heterogeneity of cartilage regeneration ability. Previous reports demonstrated that basic fibroblast growth factor (bFGF) can promote proliferation and cartilage differentiation potential of MSCs in vitro, although no reports show its beneficial effect in vivo. The purpose of this study is to investigate the promoting effect of bFGF on cartilage regeneration using human SMSC in vivo. SMSCs were cultured with or without bFGF in a growth medium, and 2 × 10⁵ cells were aggregated to form a synovial pellet. Synovial pellets were implanted into osteochondral defects induced in the femoral trochlea of severe combined immunodeficient mice, and histological evaluation was performed after eight weeks. The presence of implanted SMSCs was confirmed by the observation of human vimentin immunostaining-positive cells. Interestingly, broad lacunae structures and cartilage substrate stained by Safranin-O were observed only in the bFGF (+) group. The bFGF (+) group had significantly higher O’Driscoll scores in the cartilage repair than the bFGF (−) group. The addition of bFGF to SMSC growth culture may be a useful treatment option to promote cartilage regeneration in vivo. Full article

Methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (CS)-biofunctionalized MeHA (CS-MeHA), were crosslinked in the presence of a matrix metalloproteinase 7 (MMP7)-sensitive peptide. The synthesized hydrogels were embedded with either human mesenchymal stem cells (hMSCs) or chondrocytes, at low concentrations, and subsequently cultured in a stem cell medium (SCM) or chondrogenic induction medium (CiM). The pivotal role of the synthesized hydrogels in promoting the expression of cartilage-related genes and the formation of neocartilage tissue despite the low concentration of encapsulated cells was assessed. It was found that hMSC-laden MeHA hydrogels cultured in an expansion medium exhibited a significant increase in the expression of chondrogenic markers compared to hMSCs cultured on a tissue culture polystyrene plate (TCPS). This favorable outcome was further enhanced for hMSC-laden CS-MeHA hydrogels, indicating the positive effect of the glycosaminoglycan binding peptide on the differentiation of hMSCs towards a chondrogenic phenotype. However, it was shown that an induction medium is necessary to achieve full span chondrogenesis. Finally, the histological analysis of chondrocyte-laden MeHA hydrogels cultured on an ex vivo osteochondral platform revealed the deposition of glycosaminoglycans (GAGs) and the arrangement of chondrocyte clusters in isogenous groups, which is characteristic of hyaline cartilage morphology. Full article

Background: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Methods: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Results: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Conclusions: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs. Full article

This study aims to prepare biphasic osteochondral scaffolds based on seamless joining of sintered polymer and polymer/ceramic microspheres for co-culture of chondrocytes and bone marrow stem cells (BMSCs). Poly(lactide-co-glycolide) (PLGA) microspheres and 10% nanohydroxyapatite (nHAP)-incorporated PLGA (PGA/nHAP) microspheres were prepared through the oil-in-water precipitation method. Virgin (V) and composite (C) scaffolds were prepared from 250–500 µm PLGA and PLGA/nHAP microspheres, respectively, while osteochondral (OC) scaffolds were fabricated through the combination of V and C scaffolds. Physico-chemical properties of scaffolds were characterized through microscopic-spectroscopic evaluations. The effect of nHAP in scaffolds was investigated through thermogravimetric analysis and mechanical testing, while surface hydrophobicity was tested through contact angle measurements. Rabbit chondrocytes and BMSCs were used for cell culture, and cell morphology and proliferation were determined from SEM and DNA assays. Alizarin red and Alcian blue stains were used to identify the in vitro bone and cartilage tissue-specific regeneration, while cetylpyridinium chloride was used to quantitatively estimate calcium in mineralized bone. For co-culture in OC scaffolds, BMSCs were first seeded in the bone part of the scaffold and cultured in osteogenic medium, followed by seeding chondrocytes in the cartilage part, and cultured in chondrocyte medium. High cell viability was confirmed from the Live/Dead assays. Actin cytoskeleton organization obtained by DAPI-phalloidin staining revealed proper organization of chondrocytes and BMSCs in OC scaffolds. Immunofluorescent staining of bone (type I collagen and osteocalcin (OCN)) and cartilage marker proteins (type II collagen (COL II)) confirmed cellular behavior of osteoblasts and chondrocytes in vitro. Using an ectopic osteochondral defect model by subcutaneous implantation of co-cultured OC scaffolds in nude mice confirmed cell proliferation and tissue development from gross view and SEM observation. IF staining of OCN and COL II in the bone and cartilage parts of OC scaffolds and tissue-specific histological analysis exhibited a time-dependent tissue re-modeling and confirmed the potential application of the biphasic scaffold in osteochondral tissue engineering. Full article