Combination of a Collagen Scaffold and an Adhesive Hyaluronan-Based Hydrogel for Cartilage Regeneration: A Proof of Concept in an Ovine Model

Objective Hyaluronic acid–transglutaminase (HA-TG) is an enzymatically crosslinkable adhesive hydrogel with chondrogenic properties demonstrated in vitro and in an ectopic mouse model. In this study, we investigated the feasibility of using HA-TG in a collagen scaffold to treat chondral lesions in an ovine model, to evaluate cartilage regeneration in a mechanically and biologically challenging joint environment, and the influence of the surgical procedure on the repair process. Design Chondral defects of 6-mm diameter were created in the stifle joint of skeletally mature sheep. In a 3-month study, 6 defects were treated with HA-TG in a collagen scaffold to test the stability and biocompatibility of the defect filling. In a 6-month study, 6 sheep had 12 defects treated with HA-TG and collagen and 2 sheep had 4 untreated defects. Histologically observed quality of repair tissue and adjacent cartilage was semiquantitatively assessed. Results HA-TG adhered to the native tissue and did not cause any detectable negative reaction in the surrounding tissue. HA-TG in a collagen scaffold supported infiltration and chondrogenic differentiation of mesenchymal cells, which migrated from the subchondral bone through the calcified cartilage layer. Additionally, HA-TG and collagen treatment led to better adjacent cartilage preservation compared with empty defects ( P < 0.05). Conclusions This study demonstrates that the adhesive HA-TG hydrogel in a collagen scaffold shows good biocompatibility, supports in situ cartilage regeneration and preserves the surrounding cartilage. This proof-of-concept study shows the potential of this approach, which should be further considered in the treatment of cartilage lesions using a single-step procedure

Thermal conditioning improves quality and speed of keratinocyte sheet production for burn wound treatment

BACKGROUND AIMS Cultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes. METHODS Keratinocytes are usually grown in vitro at 37°C. Given the large temperature differences in native skin tissue, the aim of the authors' study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31°C, 33°C and 37°C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development. RESULTS The authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33°C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors. CONCLUSIONS These results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients

The role of energy dissipation of polymeric scaffolds in the mechanobiological modulation of chondrogenic expression

Mechanical stimulation has been proposed to induce chondrogenesis in cell-seeded scaffold. 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 two hours 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 scaffold with energy dissipation level close to the one of cartilage favors chondrogenic expression when dynamical loading is present

Epiphyseal chondro-progenitors provide a stable cell source for cartilage cell therapy

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 chondro-­‐progenitor (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 micro-­‐pellet 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 3 population doublings over eight 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 micro-­‐pellet format. Equally, ECPs were capable of depositing glycosaminoglycans, producing aggrecan, collagen I and collagen II in 3D pellets both at after 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 down-­‐regulation. 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

Optical fibers for endoscopic high-power Er:YAG laserosteotomy

ignificance: The highest absorption peaks of the main components of bone are in the mid-infrared region, making Er:YAG and CO2 lasers the most efficient lasers for cutting bone. Yet, studies of deep bone ablation in minimally invasive settings are very limited, as finding suitable materials for coupling high-power laser light with low attenuation beyond 2  μm is not trivial. Aim: The first aim of this study was to compare the performance of different optical fibers in terms of transmitting Er:YAG laser light with a 2.94-μm wavelength at high pulse energy close to 1 J. The second aim was to achieve deep bone ablation using the best-performing fiber, as determined by our experiments. Approach: In our study, various optical fibers with low attenuation (λ  =  2.94  μm) were used to couple the Er:YAG laser. The fibers were made of germanium oxide, sapphire, zirconium fluoride, and hollow-core silica, respectively. We compared the fibers in terms of transmission efficiency, resistance to high Er:YAG laser energy, and bending flexibility. The best-performing fiber was used to achieve deep bone ablation in a minimally invasive setting. To do this, we adapted the optimal settings for free-space deep bone ablation with an Er:YAG laser found in a previous study. Results: Three of the fibers endured energy per pulse as high as 820 mJ at a repetition rate of 10 Hz. The best-performing fiber, made of germanium oxide, provided higher transmission efficiency and greater bending flexibility than the other fibers. With an output energy of 370 mJ per pulse at 10 Hz repetition rate, we reached a cutting depth of 6.82  ±  0.99  mm in sheep bone. Histology image analysis was performed on the bone tissue adjacent to the laser ablation crater; the images did not show any structural damage. Conclusions: The findings suggest that our prototype could be used in future generations of endoscopic devices for minimally invasive laserosteotomy

Industrial Development of Standardized Fetal Progenitor Cell Therapy for Tendon Regenerative Medicine: Preliminary Safety in Xenogeneic Transplantation.

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

Development of Standardized Fetal Progenitor Cell Therapy for Cartilage Regenerative Medicine: Industrial Transposition and Preliminary Safety in Xenogeneic Transplantation

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

Industrial Development of Standardized Fetal Progenitor Cell Therapy for Tendon Regenerative Medicine: Preliminary Safety in Xenogeneic Transplantation

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

Mechanotransduction of epiphyseal chondro-progenitors in 3D hydrogel system.

Of the many external factors that affect cell behavior, mechanical cues are fundamental in modulating a cell’s phenotype. Biomimetic mechanosensing requires the three-dimensional encapsulation of cells within a controlled microenvironment. As musculoskeletal tissues are both routinely subjected and highly sensitive to mechanical cues, studying the effect of substrate properties on cellular behavior takes on a more complex angle when looked at in 3D, as is the case in vivo. This project will therefore focus on evaluating the phenotypic modulation of epiphyseal chondro-progenitors in hyaluronic acid based hydrogels both in static, free swelling culture conditions as well as the effect of dynamic unconfined compression, looking at differentially regulated cell markers which may prove fundamental for cartilage tissue regeneration

Epiphyseal Chondro-Progenitor Cell Therapy For Articular Cartilage Regeneration

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 here focuses on the reliable expansion and characterization of a clinical-grade human epiphyseal chondro-progenitor [ECP] cell bank from a single tissue donation. ECPs were investigated at both low and high cumulative population doublings, tracking morphology, monolayer expansion kinetics and resistance to cryogenic shock. Three dimensional micro-pellet assays were used to determine spontaneous cartilage-like extracellular matrix deposition. 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 and a stable population surface marker profile (CD14-, CD34-, CD45-, HLA-DP, DQ, DR-, and CD26+, CD44+, CD73+, CD90+, CD105+, CD166+, HLA-A,B,C+). ECPs also exhibit a stable spontaneous chondrogenic potential, depositing glycosaminoglycan rich matrix as well as Collagen I, Collagen II and display an inherent resistance to multilineage differentiation. ECPs were photoencapsulated in methacrylated hyaluronic acid hydrogels and subjected to dynamic compression. In response to 3D mechanostimulation, ECPs modulated the presentation of surface receptors for TGF|3, a potent chondrogenic morphogen. When co-encapsulated with bone marrow derived MSCs, the trend was reversed, pointing to potential crosstalk regulation between ECPs prone to shifting their baseline expression and the modulating MSCs. As a first step in defining a novel strategy for cartilage regeneration, we have conducted a GLP-grade pre-clinical safety study in goats to assess the effect of implanted ECPs in a full thickness cartilage defect. ECPs were delivered within a collagen-based matrix to optimize therapeutic cellular localization. The cell-laden construct is delivered in combination with microfracture to direct new tissue repair and remodeling. The results from the 3-months pre-clinical study highlight the safety of ECPs, the feasibility of the proposed treatment protocol as well as early indications as to their regenerative role and potential. The findings presented herein demonstrate the reliability, stability and responsiveness of Epiphyseal Chondro-Progenitor cells, granting them clear advantages for their use in defining novel strategies for cartilage regeneration