Exploiting Synergistic Effect of Controlled Co-Delivery Medicine for Soft Tissue Transplantation using Double System of Bio-Multifunctional Hydrogel

Autologous skin flap transplantation is a common method to repair complex soft tissue defects caused by cancer, trauma, and congenital malformations, among other conditions; however, limited blood supply range and post-transplantation ischemia-reperfusion injury (IRI) can cause distal necrosis of the flap and loss of long-term function, severely limiting the indications for this type of surgery. Here, development of a bio-multifunctional hydrogel for programmatic co-delivery medicine, DCDM, that meets the blood supply requirements of skin flap transplantation and can deliver two types of gas molecules, CO and NO, in a timely manner and with a concentration gradient during surgery, is described. DCDM maintains the immediate opening of blood flow channels in transplanted tissue and effective blood perfusion throughout the perioperative period, activating perfusion of the hemodynamic donor site. These results demonstrate that DCDM promotes distal vascularization and long-term functional reconstruction of transplanted tissues by inhibiting inflammatory damage and accelerating blood vessel formation. The findings presented here are applicable to the extended fields of large soft tissue defect repair and transplant of various organs. RNA integrity was assessed using an RNA Nano 6000 Assay Kit for the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for RNA sample preparation. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase, then RNaseH used to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTPs. Remaining overhangs were converted into blunt ends using exonuclease/polymerase activities. After adenylation of the 3' termini of DNA fragments, adaptors with hairpin loop structures were ligated to prepare for hybridization. To preferentially select cDNA fragments of 370–420 bp in length, library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified using the AMPure XP system, and library quality assessed on the Agilent Bioanalyzer 2100 system. Clustering of index-coded samples was performed using a cBot Cluster Generation System with a TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer’s instructions. After cluster generation, prepared libraries were sequenced on an Illumina Novaseq platform to generate 150 bp paired-end reads. The data obtained were subjected to quality control, reads mapped to the reference genome, novel transcripts predicted, gene expression levels quantified, differential expression analysis quantified, as well as GO and KEGG enrichment analysis, Gene Set Enrichment Analysis, single nucleotide polymorphism analysis, alternative splicing analysis, and protein-protein interaction analysis of differentially expressed genes, including weighted correlation network analysis.