Articular cartilage regeneration poses particularly tough challenges for implementing cell-based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented focuses on the reliable expansion and characterization of a clinical grade human epiphyseal chondroprogenitor (ECP) cell bank from a single tissue donation. A parental human ECP cell bank was established, which provides the seed material for master and working cell banks. ECPs were investigated at both low and high cumulative population doublings looking at morphology, monolayer expansion kinetics, resistance to cryogenic shock, colony-forming efficiency, and cell surface markers. Three-dimensional micropellet assays were used to determine spontaneous extracellular matrix deposition at varying population doublings and monolayer 2D differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential averaging three population doublings over 8 days. Surface marker analysis revealed no detectable contaminating subpopulations or population enrichment during prolonged culture periods. Despite a slight reduction in Sox9 expression levels at higher population doublings in monolayer, nuclear localization was equivalent both in monolayer and in micropellet format. Equally, ECPs were capable of depositing glycosaminoglycans and producing aggrecan, collagen I, and collagen II in 3D pellets both at low and high population doublings indicating a stable spontaneous chondrogenic potential. Osteogenic induction was differentially restricted in low and high population doublings as observed by Von Kossa staining of calcified matrix, with a notable collagen X, MMP13, and ADAMTS5 downregulation. Rare adipogenic induction was seen as evidenced by cytoplasmic lipid accumulation detectable by Oil Red O staining. These findings highlight the reliability, stability, and responsiveness of ECPs over prolonged culture, making them ideal candidates in defining novel strategies for cartilage regeneration.

a b s t r a c tMechanical stimulation has been proposed to induce chondrogenesis in cell-seeded scaffolds. However, the effects of mechanical stimuli on engineered cartilage may vary substantially between different scaffolds. This advocates for the need to identify an overarching mechanobiological variable. We hypothesize that energy dissipation of scaffolds subjected to dynamic loading may be used as a mechanobiology variable. The energy dissipation would furnish a general criterion to adjust the mechanical stimulation favoring chondrogenesis in scaffold. Epiphyseal chondro-progenitor cells were then subject to unconfined compression 2 h per day during four days in different scaffolds, which differ only by the level of dissipation they generated while keeping the same loading conditions. Scaffolds with higher dissipation levels upregulated the mRNA of chondrogenic markers. In contrast lower dissipation of scaffolds was associated with downregulation of chondrogenic markers. These results showed that energy dissipation could be considered as a mechanobiology variable in cartilage. This study also indicated that scaffolds with energy dissipation level close to the one of cartilage favors chondrogenic expression when dynamical loading is present.

Tendon defects require multimodal therapeutic management over extensive periods and incur high collateral burden with frequent functional losses. Specific cell therapies have recently been developed in parallel to surgical techniques for managing acute and degenerative tendon tissue affections, to optimally stimulate resurgence of structure and function. Cultured primary human fetal progenitor tenocytes (hFPT) have been preliminarily considered for allogeneic homologous cell therapies, and have been characterized as stable, consistent, and sustainable cell sources in vitro. Herein, optimized therapeutic cell sourcing from a single organ donation, industrial transposition of multi-tiered progenitor cell banking, and preliminary preclinical safety of an established hFPT cell source (i.e., FE002-Ten cell type) were investigated. Results underlined high robustness of FE002-Ten hFPTs and suitability for sustainable manufacturing upscaling within optimized biobanking workflows. Absence of toxicity or tumorigenicity of hFPTs was demonstrated in ovo and in vitro, respectively. Furthermore, a 6-week pilot good laboratory practice (GLP) safety study using a rabbit patellar tendon partial-thickness defect model preliminarily confirmed preclinical safety of hFPT-based standardized transplants, wherein no immune reactions, product rejection, or tumour formation were observed. Such results strengthen the rationale of the multimodal Swiss fetal progenitor cell transplantation program and prompt further investigation around such cell sources in preclinical and clinical settings for musculoskeletal regenerative medicine.

Finite element analysis with models derived from computed tomography (CT) scans is potentially powerful as a translational research tool because it can achieve what animal studies and cadaver biomechanics cannot-low-risk, noninvasive, objective assessment of outcomes in living humans who have actually experienced the injury, or treatment being studied. The purpose of this study was to assess the validity of CT-based virtual mechanical testing with respect to physical biomechanical tests in a large animal model. Three different tibial osteotomy models were performed on 44 sheep. Data from 33 operated limbs and 20 intact limbs was retrospectively analyzed. Radiographic union scoring was performed on the operated limbs and physical torsional tests were performed on all limbs. Morphometric measures and finite element models were developed from CT scans and virtual torsional tests were performed to assess healing with four material assignment techniques. In correlation analysis, morphometric measures and radiographic scores were unreliable predictors of biomechanical rigidity, while the virtual torsion test results were strongly and significantly correlated with measured biomechanical test data, with high absolute agreement. Overall, the results validated the use of virtual mechanical testing as a reliable in vivo assessment of structural bone healing. This method is readily translatable to clinical evaluation for noninvasive assessment of the healing progress of fractures with minimal risk. Clinical significance: virtual mechanical testing can be used to reliably and noninvasively assess the rigidity of a healing fracture using clinical-resolution CT scans and that this measure is superior to morphometric and radiographic measures.

BackgroundWound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC + K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five.ResultsHealing time was reduced from 13.9 ± 0.5 days (mean ± SEM) in the control group to 7.2 ± 0.2 days in the PC group (P < 0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7 ± 0.2 days. Pain was reduced in both the PC and PC + K groups. Data showed a statistically detectable advantage of using PC + K over PC alone (P < 0.01).ConclusionThe results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence.Clinical trial registry informationProtocol Record Identification Number: 132/03Registry URL: http://www.clinicaltrials.gov

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Diverse cell therapy approaches constitute prime developmental prospects for managing acute or degenerative cartilaginous tissue affections, synergistically complementing specific surgical solutions. Bone marrow stimulation (i.e., microfracture) remains a standard technique for cartilage repair promotion, despite incurring the adverse generation of fibrocartilagenous scar tissue, while matrix-induced autologous chondrocyte implantation (MACI) and alternative autologous cell-based approaches may partly circumvent this effect. Autologous chondrocytes remain standard cell sources, yet arrays of alternative therapeutic biologicals present great potential for regenerative medicine. Cultured human epiphyseal chondro-progenitors (hECP) were proposed as sustainable, safe, and stable candidates for chaperoning cartilage repair or regeneration. This study describes the development and industrial transposition of hECP multi-tiered cell banking following a single organ donation, as well as preliminary preclinical hECP safety. Optimized cell banking workflows were proposed, potentially generating millions of safe and sustainable therapeutic products. Furthermore, clinical hECP doses were characterized as non-toxic in a standardized chorioallantoic membrane model. Lastly, a MACI-like protocol, including hECPs, was applied in a three-month GLP pilot safety evaluation in a caprine model of full-thickness articular cartilage defect. The safety of hECP transplantation was highlighted in xenogeneic settings, along with confirmed needs for optimal cell delivery vehicles and implantation techniques favoring effective cartilage repair or regeneration.

Nasal chondrocyte–based grafts modulate osteoarthritis-like conditions in animal models and improve life quality in patients with knee osteoarthritis.