Cartilage Defect Treatment Using High-Density Autologous Chondrocyte Implantation (HD-ACI)

Guillén-García, Pedro; Guillén-Vicente, Isabel; Rodríguez-Iñigo, Elena; Guillén-Vicente, Marta; Fernández-Jaén, Tomás Fernando; Navarro, Ramón; Aboli, Lucía; Torres, Raúl; Abelow, Steve; López-Alcorocho, Juan Manuel

doi:10.3390/bioengineering10091083

Cited by 8 publications

(4 citation statements)

References 55 publications

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“…The capacity of PRF to provide a similar effect to 20% FBS for the in vitro cultivation of pOBs was recently demonstrated [ 35 ], further supporting the results of the present study. Our effects observed in vitro are of high value for cell therapy procedures, in which cells are harvested from patients, cultivated in vitro, and reimplanted into the same patient as a type of autologous transplantation [ 36 ]. In this procedure, the use of PRF from the same patient instead of external FBS would greatly help keep this autologous system closed without external additives.…”

Section: Discussionmentioning

confidence: 99%

Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study

Al-Maawi,

Dohle,

Sader

et al. 2024

Bioengineering

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Platelet-rich fibrin (PRF) has assumed an important role in supporting tissue regeneration in different fields. To date, the standard protocol for liquid PRF requires at least 10 mL of peripheral blood. The present study aimed to analyze the composition, growth factor release, and effects on the cell proliferation of PRF samples produced using 3 mL vs. 10 mL of peripheral blood in vitro. Peripheral venous blood from six healthy donors was used to prepare liquid PRF using either 3 mL or 10 mL tubes. Three different centrifugation protocols were used according to the low-speed centrifugation concept. The cellular distribution was evaluated using immunohistology and automated cell count. ELISA was used to determine the release of different growth factors (EGF, TGF-β1, and PDGF) and interleukin 8 at different time points. Primary human osteoblasts (pOBs) were cultivated for 7 days using PRF-conditioned media acquired from either 3 mL or 10 mL of peripheral blood. The results showed that 3 mL of peripheral blood is sufficient to produce a liquid PRF concentrate similar to that acquired when using 10 mL blood. The concentrations of platelets and leukocytes were comparable regardless of the initial blood volume (3 mL vs. 10 mL). Similarly, the release of growth factors (EGF, TGF-β1, and PDGF) and interleukin 8 was often comparable in both groups over 7 days. The cultivation of pOBs using PRF-conditioned media showed a similar proliferation rate regardless of the initial blood volume. This proliferation rate was also similar to that of pOBs treated with 20% FBS-conditioned media. These findings validated the use of 3 mL of peripheral blood to generate liquid PRF matrices according to the low-speed centrifugation concept, which may open new application fields for research purposes such as in vivo experiments and clinical applications such as pediatric surgery.

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Section: Discussionmentioning

confidence: 99%

Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study

Al-Maawi,

Dohle,

Sader

et al. 2024

Bioengineering

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“…This not only further strengthens the findings previously reported by our group [ 29 ], but radically expands the impact of bulk chondrocytes gene editing to potentially develop not only allogeneic therapies (with e.g., clinical grade FE002 chondroprogenitor cells and polydactyly chondrocytes), but also, for the first time, autologous gene therapy strategies with patients-derived OA chondrocytes. Autologous chondrocytes implantation (ACI) and matrix-induced ACI (MACI) are two cell-based therapies for cartilage regeneration that rely on the harvesting of a small biopsy, followed by autologous chondrocytes culturing ex vivo and subsequent re-implantation into the site of the lesion [ 59 ]. Despite being a commercially available product [ 60 ], ACI and MACI carry significant limitations.…”

Section: Discussionmentioning

confidence: 99%

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Ponta,

Bonato,

Neidenbach

et al. 2024

Arthritis Res Ther

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Background CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources. Methods We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β). Results We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions. Conclusions We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

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“…Chondrocytes are grown, either in vivo or in vitro, until millions of cells are obtained. Cultured chondrocytes are then implanted into the damaged area of articular cartilage as a patch [ 10 ]. ACI can provide an alternative cell resource with a higher regenerative capacity for cartilage regeneration.…”

Section: Development Of Autologous Chondrocyte Implantationmentioning

confidence: 99%

Latest Advances in Chondrocyte-Based Cartilage Repair

Yue,

Lim,

Owens

2024

Biomedicines

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Chondrocyte-based cell therapy has been used for more than 30 years and is still considered to be a promising method of cartilage repair despite some limitations. This review introduces the latest developments of four generations of autologous chondrocyte implantation and current autologous chondrocyte products. The regeneration of cartilage from adult chondrocytes is limited by culture-induced dedifferentiation and patient age. Cartibeads is an innovative three-step method to produce high-quality hyaline cartilage microtissues, and it is developed from adult dedifferentiated chondrocytes with a high number of cell passages. In addition, allogeneic chondrocyte therapies using the Quantum hollow-fiber bioreactor and several signaling pathways involved in chondrocyte-based cartilage repair are mentioned, such as WNT signaling, the BMP-2/WISP1 pathway, and the FGF19 pathway.

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Cartilage Defect Treatment Using High-Density Autologous Chondrocyte Implantation (HD-ACI)

Cited by 8 publications

References 55 publications

Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study

Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Latest Advances in Chondrocyte-Based Cartilage Repair

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