Ex Vivo Functional Benchmarking of Hyaluronan-Based Osteoarthritis Viscosupplement Products: Comprehensive Assessment of Rheological, Lubricative, Adhesive, and Stability Attributes.

While many injectable viscosupplementation products are available for osteoarthritis (OA) management, multiple hydrogel functional attributes may be further optimized for efficacy enhancement. The objective of this study was to functionally benchmark four commercially available hyaluronan-based viscosupplements (Ostenil, Ostenil Plus, Synvisc, and Innoryos), focusing on critical (rheological, lubricative, adhesive, and stability) attributes. Therefore, in vitro and ex vivo quantitative characterization panels (oscillatory rheology, rotational tribology, and texture analysis with bovine cartilage) were used for hydrogel product functional benchmarking, using equine synovial fluid as a biological control. Specifically, the retained experimental methodology enabled the authors to robustly assess and discuss various functional enhancement options for hyaluronan-based hydrogels (chemical cross-linking and addition of antioxidant stabilizing agents). The results showed that the Innoryos product, a niacinamide-augmented linear hyaluronan-based hydrogel, presented the best overall functional behavior in the retained experimental settings (high adhesivity and lubricity and substantial resistance to oxidative degradation). The Ostenil product was conversely shown to present less desirable functional properties for viscosupplementation compared to the other investigated products. Generally, this study confirmed the high importance of formulation development and control methodology optimization, aiming for the enhancement of novel OA-targeting product critical functional attributes and the probability of their clinical success. Overall, this work confirmed the tangible need for a comprehensive approach to hyaluronan-based viscosupplementation product functional benchmarking (product development and product selection by orthopedists) to maximize the chances of effective clinical OA management.

Autologous and Allogeneic Cytotherapies for Large Knee (Osteo)Chondral Defects: Manufacturing Process Benchmarking and Parallel Functional Qualification.

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.

[Treatment of extensive chondral and osteochondral knee defects with autologous chondrocytes implantation]. / Grands défects chondraux et ostéochondraux du genou : traitement par greffe chondrocytaire autologue.

Treatment of extensive chondral and osteochondral defects in the knee remains a challenge. The traditional bone marrow stimulation and osteochondral mosaicplasty are effective but this is indicated only for lesions smaller than 4 cm2. In addition, while they are offered in young patients, functional deterioration is often observed after 2 years. In contrast, Autologous Chondrocyte Implantation (ACI) has proven to be efficient and durable even in larger lesions. These factors have encouraged our team to consider ACI as a valuable tool, but it was not readily available in Switzerland. In this article, we describe why and how we have developed and refined the technique in our University Hospital for the clinical implementation of this cell-based therapy, performed under a quality assurance system and following good manufacturing practices.

Dans le traitement des défects chondraux et ostéochondraux du genou, les techniques classiques de réparation par stimulation de la moelle osseuse et de greffe ostéochondrale autologue (mosaicplastie) présentent d'importantes limites quant à la taille des lésions traitables (< 4 cm2, soit < 22 mm de diamètre). De plus, alors qu'elles sont destinées à des patients jeunes, une dégradation des résultats a été rapportée après 2 ans. Ces éléments nous ont incités à nous orienter vers la greffe chondrocytaire autologue afin de pouvoir proposer aux patients le traitement de lésions plus étendues avec une meilleure durabilité. Cet article décrit pourquoi et comment nous avons implémenté cette voie thérapeutique dans notre centre universitaire, en assurant une traçabilité et des contrôles qualités stricts tout au long de la chaîne de production.

Retrospective Analysis of Autologous Chondrocyte-Based Cytotherapy Production for Clinical Use: GMP Process-Based Manufacturing Optimization in a Swiss University Hospital.

Cultured autologous human articular chondrocyte (HAC) implantation has been extensively investigated for safe and effective promotion of structural and functional restoration of knee cartilage lesions. HAC-based cytotherapeutic products for clinical use must be manufactured under an appropriate quality assurance system and follow good manufacturing practices (GMP). A prospective clinical trial is ongoing in the Lausanne University Hospital, where the HAC manufacturing processes have been implemented internally. Following laboratory development and in-house GMP transposition of HAC cell therapy manufacturing, a total of 47 patients have been treated to date. The main aim of the present study was to retrospectively analyze the available manufacturing records of the produced HAC-based cytotherapeutic products, outlining the inter-individual variability existing among the 47 patients regarding standardized transplant product preparation. These data were used to ameliorate and to ensure the continued high quality of cytotherapeutic care in view of further clinical investigations, based on the synthetic analyses of existing GMP records. Therefore, a renewed risk analysis-based process definition was performed, with specific focus set on process parameters, controls, targets, and acceptance criteria. Overall, high importance of the interdisciplinary collaboration and of the manufacturing process robustness was underlined, considering the high variability (i.e., quantitative, functional) existing between the treated patients and between the derived primary HAC cell types.

Human Platelet Lysate as an Alternative to Autologous Serum for Human Chondrocyte Clinical Use.

OBJECTIVE: A pivotal aspect of cartilage tissue engineering resides in cell culture medium supplementation, in view of maximizing in vitro cell proliferation and preserving cellular functionality. Autologous human serum (aHS) is commonly used as an inducive supplement for safe human articular chondrocyte (HAC) proliferation prior to clinical implantation. However, practical clinical use of aHS is hindered by constraining manufacturing requirements and quality assurance-driven downstream processing. The present study investigated potential alternative use of commercial human platelet lysate (hPL) supplements in HAC manufacturing workflows related to clinical therapeutic pathways. DESIGN: Differential effects of hPL, aHS, and fetal bovine serum were assessed on primary cultured HAC parameters (viability, proliferative rates, and morphology) in 2-dimensional in vitro systems. A 3-dimensional HAC pellet model served for postexpansion assessment of cellular functionality, by visualizing proteoglycan production (Alcian blue staining), and by using qRT-PCR relative quantification of chondrogenic marker (SOX9, COL2-A1, and ACAN) genetic expression. RESULTS: We found that monolayer HAC culture with hPL or aHS supplements presented similar characteristics (elongated cell morphology and nearly identical growth kinetics). Chondrogenic activity appeared as conserved in HACs expanded with human or bovine supplements, wherein histologic analysis indicated a progressive sGAG accumulation and SOX9, COL2-A1, ACAN gene expression was upregulated in 3-dimensional HAC pellet models. CONCLUSION: This study therefore supports the use of hPL as a functional equivalent and alternative to aHS for cultured HAC batch preparation, with the potential to effectively alleviate pressure on clinical and manufacturing bottlenecks in cell therapy approaches for cartilage regeneration.

Antagonistic effect of the inflammasome on thymic stromal lymphopoietin expression in the skin.

BACKGROUND: Innate immune sensors control key cytokines that regulate T-cell priming and T-cell fate. This is particularly evident in allergic reactions, which represent ideal systems to study the interplay of innate and adaptive immunity. In patients with contact dermatitis, inflammasome-mediated IL-1 activation is responsible for a TH1 immune response. Surprisingly, the IL-1 signaling pathway was also proposed to control the activation of thymic stromal lymphopoietin (TSLP), a cytokine implicated in development of the T(H)2 response in patients with atopic dermatitis (AD) and asthma. OBJECTIVES: We sought to assess the effect of the inflammasome on TSLP expression levels and the development of AD. METHODS: We studied the effect of the inflammasome activator 2,4-dinitrofluorobenzene, and IL-1ß on TSLP mRNA expression levels in mouse and human cell lines (in vitro assays), as well as in live mice and on human skin transplants. We also assessed the effect of 2,4-dinitrofluorobenzene on TSLP and the TH2 response in mice in which the inflammasome and IL-1 signaling pathways were blocked, either genetically or pharmacologically, in 2 models of AD. RESULTS: We provide in vitro and in vivo evidence that inflammasome activation has an inhibitory role on TSLP mRNA expression and T(H)2 cell fate in the skin. We also show that solvents influence the activation of TSLP and IL-1ß and direct the T-cell fate to a given hapten. CONCLUSION: Our observations strongly suggest that the TH1 versus TH2 cell fate decision is regulated at multiple levels and starts with innate immune events occurring within peripheral epithelial tissues.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.