Activity

The development of alternatives for autologous bone grafts is a major focus of bone tissue engineering. To produce living bone-forming implants, skeletal stem and progenitor cells (SSPCs) are envisioned as key ingredients. SSPCs can be obtained from different tissues including bone marrow, adipose tissue, dental pulp, and periosteum. Human periosteum-derived cells (hPDCs) exhibit progenitor cell characteristics and have well-documented in vivo bone formation potency. Selleck CCT128930 Here, we have characterized and compared hPDCs derived from tibia with craniofacial hPDCs, from maxilla and mandible, respectively, each representing a potential source for cell-based tissue engineered implants for craniofacial applications. Maxilla and mandible-derived hPDCs display similar growth curves as tibial hPDCs, with equal trilineage differentiation potential toward chondrogenic, osteogenic, and adipogenic cells. These craniofacial hPDCs are positive for SSPC-markers CD73, CD164, and Podoplanin (PDPN), and negative for CD146, hematopoietic and endothelial lineage markers. Bulk RNA-sequencing identified genes that are differentially expressed between the three sources of hPDC. In particular, differential expression was found for genes of the HOX and DLX family, for SOX9 and genes involved in skeletal system development. The in vivo bone formation, 8 weeks after ectopic implantation in nude mice, was observed in constructs seeded with tibial and mandibular hPDCs. Taken together, we provide evidence that hPDCs show different profiles and properties according to their anatomical origin, and that craniofacial hPDCs are potential sources for cell-based bone tissue engineering strategies. The mandible-derived hPDCs display – both in vitro and in vivo – chondrogenic and osteogenic differentiation potential, which supports their future testing for use in craniofacial bone regeneration applications.The H19 gene promotes skeletal muscle differentiation in mice, but the regulatory models and mechanisms of myogenesis regulated by H19 are largely unknown in pigs. Therefore, the regulatory modes of H19 in the differentiation of porcine skeletal muscle satellite cells (PSCs) need to be determined. We observed that H19 gene silencing could decrease the expressions of the myogenin (MYOG) gene, myogenic differentiation (MYOD), and myosin heavy chain (MYHC) in PSCs. Therefore, we constructed and sequenced 12 cDNA libraries of PSCs after knockdown of H19 at two differentiation time points to analyze the transcriptome differences. A total of 11,419 differentially expressed genes (DEGs) were identified. Among these DEGs, we found through bioinformatics analysis and protein interaction experiment that SRY-box transcription factor 4 (SOX4) and Drebrin 1 (DBN1) were the key genes in H19-regulated PSC differentiation. Functional analysis shows that SOX4 and DBN1 promote PSC differentiation. Mechanistically, H19 regulates PSC differentiation through two different pathways. On the one hand, H19 functions as a molecular sponge of miR-140-5p, which inhibits the differentiation of PSCs, thereby modulating the derepression of SOX4. On the other hand, H19 regulates PSC differentiation through directly binding with DBN1. Furthermore, MYOD binds to the promoters of H19 and DBN1. The knockdown of MYOD inhibits the expression of H19 and DBN1. We determined the function of H19 and provided a molecular model to elucidate H19’s role in regulating PSC differentiation.Gelatin methacryloyl (GelMA) has been widely used in bone engineering. It can also be filled into the calvarial defects with irregular shape. However, lack of osteoinductive capacity limits its potential as a candidate repair material for calvarial defects. In this study, we developed an injectable magnesium-zinc alloy containing hydrogel complex (Mg-IHC), in which the alloy was fabricated in an atomization process and had small sphere, regular shape, and good fluidity. Mg-IHC can be injected and plastically shaped. After cross-linking, it contents the elastic modulus similar to GelMA, and has inner holes suitable for nutrient transportation. Furthermore, Mg-IHC showed promising biocompatibility according to our evaluations of its cell adhesion, growth status, and proliferating activity. The results of alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, and real-time polymerase chain reaction (PCR) further indicated that Mg-IHC could significantly promote the osteogenic differentiation of MC3T3-E1 cells and upregulate the genetic expression of collagen I (COL-I), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2). Finally, after applied to a mouse model of critical-sized calvarial defect, Mg-IHC remarkably enhanced bone formation at the defect site. All of these results suggest that Mg-IHC can promote bone regeneration and can be potentially considered as a candidate for calvarial defect repairing.The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS.