METHODS: This is a dual-center randomized controlled trial (RCT). Sixty-nine patients aged 18 to 55 years with International Cartilage Repair Society grade 3 and 4 chondral lesions (size ≥3 cm2) of the knee joint were randomized equally into (1) a control group receiving intra-articular injections of HA plus physiotherapy and (2) an intervention group receiving arthroscopic subchondral drilling into chondral defects and postoperative intra-articular injections of PBSCs plus HA. The coprimary efficacy endpoints were subjective International Knee Documentation Committee (IKDC) and Knee Injury and Osteoarthritis Outcome Score (KOOS)-pain subdomain measured at month 24. The secondary efficacy endpoints included all other KOOS subdomains, Numeric Rating Scale (NRS), and Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scores.
RESULTS: At 24 months, the mean IKDC scores for the control and intervention groups were 48.1 and 65.6, respectively (P < .0001). The mean for KOOS-pain subdomain scores were 59.0 (control) and 86.0 (intervention) with P < .0001. All other KOOS subdomain, NRS, and MOCART scores were statistically significant (P < .0001) at month 24. Moreover, for the intervention group, 70.8% of patients had IKDC and KOOS-pain subdomain scores exceeding the minimal clinically important difference values, indicating clinical significance. There were no notable adverse events that were unexpected and related to the study drug or procedures.
CONCLUSIONS: Arthroscopic marrow stimulation with subchondral drilling into massive chondral defects of the knee joint followed by postoperative intra-articular injections of autologous PBSCs plus HA is safe and showed a significant improvement of clinical and radiologic scores compared with HA plus physiotherapy.
LEVEL OF EVIDENCE: Level I, RCT.
MATERIALS AND METHODS: The differentiation of fibroblast-like cells from SHED was carried out by using specific human recombinant connective tissue growth factor (CTGF). To characterize fibroblastic differentiation, the induced cells were subjected to morphological changes, proliferation rate, gene expression analysis using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and immunofluorescence staining. The commercial primary human gingival fibroblasts served as positive control in this study.
RESULTS: The results from characterization analysis were compared with that of commercial cells to ensure that the cells differentiated from SHED were fibroblast-like cells. The results showed the inductive effect of CTGF for fibroblastic differentiation in SHED. SHED-derived fibroblasts were successfully characterized despite having similar morphological appearance, i.e., (i) significant proliferation rate between fibroblast-like cells and SHED, (ii) high expression of fibroblast-associated markers in qRT-PCR analysis, and (iii) positive staining against collagen type 1, fibroblast-specific protein 1, and human thymic fibroblasts in flow cytometry analysis and immunofluorescence staining. The same expression patterns were found in primary human gingival fibroblasts, respectively. SHED as negative control showed lower expression or no signal, thus confirming the cells differentiated from SHED were fibroblast-like cells.
CONCLUSIONS: Taken together, the protocol adopted in this study suggests CTGF to be an appropriate inducer in the differentiation of SHED into fibroblast-like cells.
CLINICAL RELEVANCE: The fibroblast-like cells differentiated from SHED could be used in future in vitro and in vivo dental tissue regeneration studies as well as in clinical applications where these cells are needed.
METHODS: Ten healthy subjects (aged 19-44 years) received 3 consecutive daily doses of filgrastim followed by an apheresis harvest of mononuclear cells on a fourth day. In a clean room, the apheresis product was prepared for cryopreservation and processed into 4 mL aliquots. Sterility and qualification testing were performed pre-processing and post-processing at multiple time points out to 2 years. Eight samples were shipped internationally to validate cell transport potential. One sample from all participants was cultured to test proliferative potential with colony forming unit (CFU) assay. Five samples, from 5 participants were tested for differentiation potential, including chondrogenic, adipogenic, osteogenic, endoderm, and ectoderm assays.
RESULTS: Fresh aliquots contained an average of 532.9 ± 166. × 106 total viable cells/4 mL vial and 2.1 ± 1.0 × 106 CD34+ cells/4 mL vial. After processing for cryopreservation, the average cell count decreased to 331.3 ± 79. × 106 total viable cells /4 mL vial and 1.5 ± 0.7 × 106 CD34+ cells/4 mL vial CD34+ cells. Preprocessing viability averaged 99% and postprocessing 88%. Viability remained constant after cryopreservation at all subsequent time points. All sterility testing was negative. All samples showed proliferative potential, with average CFU count 301.4 ± 63.9. All samples were pluripotent.
CONCLUSIONS: Peripheral blood stem cells are pluripotent and can be safely harvested/stored with filgrastim, apheresis, clean-room processing, and cryopreservation. These cells can be stored for 2 years and shipped without loss of viability.
CLINICAL RELEVANCE: This method represents an accessible stem cell therapy in development to augment cartilage repair.
MATERIALS AND METHODS: An electronic search was undertaken using combination of keywords e.g. Homeobox genes, tooth development, dental diseases, stem cells, induced pluripotent stem cells, gene control region was used as search terms in PubMed and Web of Science and relevant full text articles and abstract were retrieved that were written in English. A manual hand search in text books were also carried out. Articles related to homeobox genes in dentistry and tissue engineering and regenerative medicine of odontogenesis were selected.
RESULTS: The possible perspective of stem cells technology in odontogenesis and subsequent analysis of gene correction pertaining to dental disorders through the possibility of induced pluripotent stem cells technology is also inferred.
CONCLUSIONS: We demonstrate the promising role of tissue engineering and regenerative medicine on odontogenesis, which can generate a new ray of hope in the field of dental science.
METHODS: Spheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.
RESULTS: CircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.
CONCLUSIONS: CircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.
CASE PRESENTATION: Five patients with OCDs of the knee joint are presented. The etiology includes osteochondritis dissecans, traumatic knee injuries, previously failed cartilage repair procedures involving microfractures and OATS (osteochondral allograft transfer systems). PBSC were harvested 1 week after surgery. Patients received intra-articular injections at week 1, 2, 3, 4, and 5 after surgery. Then at 6 months after surgery, intra-articular injections were administered at a weekly interval for 3 consecutive weeks. These 3 weekly injections were repeated at 12, 18 and 24 months after surgery. Each patient received a total of 17 injections. Subjective International Knee Documentation Committee (IKDC) scores and MRI scans were obtained preoperatively and postoperatively at serial visits. At follow-ups of >5 years, the mean preoperative and postoperative IKDC scores were 47.2 and 80.7 respectively (p = 0.005). IKDC scores for all patients exceeded the minimal clinically important difference values of 8.3, indicating clinical significance. Serial MRI scans charted the repair and regeneration of the OCDs with evidence of bone growth filling-in the base of the defects, followed by reformation of the subchondral bone plate and regeneration of the overlying articular cartilage.
CONCLUSION: These case studies showed that this treatment is able to repair and regenerate both the osseous and articular cartilage components of knee OCDs.