Search

Purpose: The longevity of biomedical implants is limited by adverse implant-tissue interactions leading to implant failure. In almost all cases, implant failure occurs due to a phenomenon known as the foreign body response (FBR). FBR is characterized by fibrous capsule formation around implanted devices, leading to implant malfunction as well as distortion of the surrounding tissue. In the context of breast implants, FBR leads to capsular contracture, which is the cause of a significant proportion of all breast implant failures. Despite the high prevalence of FBR-mediated implant failure, the underlying mechanisms of FBR are incompletely understood. Our work utilizing mass spectrometry has demonstrated that when compared to healthy subcutaneous tissue, foreign body capsules in mice and humans display an up-regulation of IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), a scaffolding protein involved in multiple mechanotransduction pathways. Based on these findings, we sought to investigate which cellular subpopulations express IQGAP1 as well as its role in mechanotransduction pathways mediating the development of FBR. Methods: To verify the importance of IQGAP1-mediated signaling in FBR, we employed a murine model of mechanically stimulated silicone implants (MSI), which were implanted subcutaneously in wildtype (WT) and IQGAP1+/- haplo-insufficient mice to compare the effect of human levels of mechanical stress on FBR. Homozygous IQGAP1 KO mice have been previously reported to harbor a fragile phenotype, more prone to pulmonary vascular damage and gastric pathologies. Haplo-insufficient mice were utilized in these experiments in order to prevent potential systemic complications from elevated levels of mechanical stimulation produced by the MSI model. We explanted the foreign body capsules from IQGAP1+/- haplo-insufficient and WT mice and performed single-cell RNA sequencing (sc-RNA-seq) on cells isolated from the capsules. We histologically assessed the quantity as well as maturity of collagen deposition using Masson’s Trichrome and Herovici staining of tissue sections from explanted foreign body capsules. Moreover, immunostaining for the mechanotransduction related proteins alpha-smooth muscle actin (α-SMA), phosphorylated focal adhesion kinase (p-FAK), phosphorylated cell division control protein 42 (p-cdc42), and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) was performed to assess the impact of IQGAP1-deficiency on the protein level. Results: We found that IQGAP1-deficient mice displayed a significantly reduced FBR as evidenced by thinner capsules, lower levels of collagen deposition, collagen maturity, and myofibroblast activation. Our scRNA-seq analysis revealed a depletion of mechanoresponsive myeloid cells in IQGAP1-deficient mice. This was confirmed on the protein level by a significantly reduced expression of a-SMA, p-FAK, p-cdc42, and p-ERK1/2 in foreign body capsule tissue from IQGAP1-deficient mice compared to WT mice. Conclusion: Our results highlight the important role of IQGAP1 as a critical early stage mediator of mechanotransduction pathways contributing to the development of FBR. Further, we show that IQGAP1 plays a role in modulating the innate immune response to synthetic implants. Therefore, IQGAP1 may be a promising target for the development of novel therapeutics to limit the development of FBR around biomedical implants.

Purpose: Dendritic cells (DCs) are a heterogeneous cell population which critically regulates the adaptive immune response. Depending on their activation status, DCs can also promote peripheral immune tolerance, thus limiting the activation of the immune system and tissue damage. The N-myc downregulated gene 2 (Ndrg2) is highly expressed in DCs and limits the secretion of vascular endothelial growth factor (VEGF), which is critical for wound healing. Cell based therapy approaches using DCs have been approved by the FDA and clinical trials using DC immunotherapy are being performed against a variety of cancer types. However, the role of DC therapy for wound healing has not yet been investigated. Methods: Hematopoietic progenitor cells were isolated from the bone marrow of mice and differentiated into DCs over a 7-day in vitro culture period. Pharmacologic down-regulation of Ndrg2 was performed by treatment of cultures with 1,25-dihydroxyvitamin D3 (VD3) and the angiogenic potential of the treated cells was evaluated by endothelial cell (EC) tube formation assays. Cytokine secretion of DCs was measured in the conditioned media using Luminex multiplex assays. To permanently knock out Ndrg2 in DCs, a CRISPR/Cas9 gene editing approach was developed, using Cas9/sgRNA-ribonucleoproteins and electroporation. The determine the impact of genetically edited DCs on wound healing, splinted excisional wounds in C57BL6/J (wild-type) mice were treated weekly with pullulan-collagen hydrogels seeded with Ndrg2-knockout (KO) DCs, control DCs which had undergone electroporation only, or blank hydrogels. The transcriptomic impact of Ndrg2 downregulation on DC fate was evaluated by microfluidic single-cell RNA sequencing (scRNA seq) of Ndrg2-KO DCs, VD3-treated DCs and control DCs. Results: Ndrg2 down-regulation lead to a significantly stronger EC tube formation in co-cultures with VD3-treated DCs, and strongly enhanced VEGF secretion compared to untreated DCs in vitro. A CRISPR/Cas9 editing pipeline was developed for KO of Ndrg2 in DCs with a transfection rate and editing efficiency of > 90% shown by Sanger Sequencing. Excisional wounds treated with Ndrg2-KO DCs demonstrated significantly accelerated healing compared to control DCs and blank hydrogels. scRNA seq revealed that Ndrg2 downregulation strongly induced Vegfa expression and anti-oxidant transcriptomic signatures. Conclusion: Our data indicate that KO of Ndrg2 in DCs strongly enhances their secretion of VEGF, thus promoting angiogenesis and accelerating wound healing. Given the ready availability of DCs from the human blood through established leukapheresis protocols and easy multiplication in vitro, CRISPR/Cas9 editing of DCs is a promising new approach to induce wound healing and soft-tissue regeneration.