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

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-1ß (IL-1ß). 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-1ß 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.

Chondrocytes From Device-Minced Articular Cartilage Show Potent Outgrowth Into Fibrin and Collagen Hydrogels.

BACKGROUND: Transplantation of autologous minced cartilage is an established procedure to repair chondral lesions. It relies on the migration of chondrocytes out of cartilage particles into a biomaterial. So far, there is no efficient way to finely mince cartilage. No consensus exists on the nature of the biomaterial to be used to promote chondrocyte migration. PURPOSE/HYPOTHESIS: This study aimed to investigate the potential clinical use of a custom-made mincing device as well as a possible alternative biomaterial to fibrin glue. The device was tested for its effect on chondrocyte viability and on subsequent chondrocyte migration into either a fibrin or a collagen gel. We hypothesized that device mincing would allow finer cutting and consequently more cell migration and that the gelation mechanism of the collagen biomaterial, which uses the clotting of platelet-rich plasma, would enhance matrix production by outgrown chondrocytes. STUDY DESIGN: Controlled laboratory study. METHODS: Cartilage from 12 patients undergoing knee arthroplasty was taken from the femoral condyles and subsequently either hand minced or device minced. The viability and the degree of outgrowth were quantified with live/dead assay on the generated cartilage particles and on the gels in which these particles were embedded, respectively. Matrix deposition in the biomaterials by the outgrown cells was investigated with histology. RESULTS: The device allowed rapid mincing of the cartilage and produced significantly smaller pieces than hand mincing. The initial chondrocyte viability in cartilage particles dropped by 25% with device mincing as compared with no mincing. However, the viability in hand-minced, device-minced, and unminced samples was no longer different after 7 and 28 days in culture. Outgrowth scores were similar among the 3 groups. Fibrin and collagen biomaterials equally supported chondrocyte outgrowth and survival, but neither promoted matrix deposition after in vitro culture. CONCLUSION: The outgrowth potential, the viability after 28 days in culture, and the matrix deposition were not different between the mincing techniques and the tested biomaterials, yet device mincing is faster and results in significantly smaller cartilage particles. CLINICAL RELEVANCE: Device mincing could become the standard method to mince cartilage for second-generation cartilage repair techniques.

Functional outcomes after nonoperative management of fractures of the proximal humerus.

BACKGROUND: Prospective follow-up data after nonoperative treatment for fractures of the proximal humerus are scarce. We studied functional outcomes and rates of complication and failure after conservative management of these common injuries. MATERIALS AND METHODS: Consecutive patients aged older than 18 years presenting to the emergency department of a large district hospital with an isolated, closed proximal humeral fracture considered suitable for functional treatment by the surgeon on charge were enrolled in a prospective, externally monitored observational study. Surgeons were free to reduce the fracture and to prescribe any type of sling or brace. Active follow-up after 12 weeks, 6, and 12 months included plain radiographs, Constant score, and Disabilities of Arm, Shoulder and Hand (DASH) score. RESULTS: We enrolled 160 patients (118 women; mean age, 63.3 +/- 14.8 years), and 124 completed 1-year follow-up. There were 85, 71, and 4 AO 11 A, B, and C fractures, and 75 one-part, 60 two-part, 23 three-part, and 2 four-part and head-splitting fractures. After 1 year, the mean difference in Constant scores between the injured and contralateral shoulder was 8.2 (95% confidence interval [CI], 6.0-10.4). The mean difference in 1-year DASH scores to baseline assessment was 10.2 points (95% CI 7.3-13.1 points). The risk of delayed and nonunion was 7.0% (95% CI, 3.6%-12.3%). Four patients subsequently underwent surgical fixation, and 5 had arthroscopic subacromial decompression. CONCLUSION: This study may provide reference values for future investigations and stresses ceiling effects that will make it difficult to demonstrate a significant advantage of surgical over nonoperative treatment in patients with proximal humeral fractures. LEVEL OF EVIDENCE: Level 4; Prospective case series without a control group.