Faculty Bibliography

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

BACKGROUND:Breast cancer resulting from a genetic mutations, such as BRCA1 or BRCA2, is seen in 5 to 10 percent of patients. More widespread genetic testing has increased the number of affected women undergoing prophylactic mastectomy and oophorectomy. Recent studies have yielded mixed results regarding complication rates after combined breast and ovarian operations. The authors compared surgical outcomes of breast operations performed in combination with salpingo-oophorectomies or as separate procedures. METHODS:The authors retrospectively analyzed surgical complications and length of hospital stay in 145 female patients, from which 87 had undergone combined breast surgery and salpingo-oophorectomy, and 58 had undergone these procedures separately. Multivariate logistic regression models were used to calculate odds ratios and 95 percent confidence intervals. RESULTS:Patients undergoing combined breast and ovarian operations experienced higher rates of overall complications (46.5 percent versus 19 percent; p < 0.001), infections (22.2 percent versus 8.6 percent; p < 0.05), and delayed wound healing (13.2 percent versus 0 percent; p < 0.05) related to the breast surgery, when compared with patients undergoing separate procedures. Multivariate logistic regression analysis confirmed a significant association between combined surgery and overall postoperative complications (OR, 5.87; 95 percent CI, 2.03 to 16.91; p = 0.02). Patients undergoing tissue expander-based breast reconstruction combined with ovarian surgery had significantly longer hospital stays compared to patients undergoing separate procedures (3.5 days versus 1.8 days; p < 0.001). CONCLUSIONS:The authors' data indicate that combining breast and ovarian operations is associated with a higher risk of postoperative complications related to the breast procedure and increases the duration of hospital stay in patients with tissue expander-based reconstructions. The authors' study provides valuable information for preoperative counseling of patients considering both breast and ovarian surgery. CLINICAL QUESTION/LEVEL OF EVIDENCE:Therapeutic, III.

Textured breast implants are associated with prolonged inflammation leading to increased risk for complications such as the development of anaplastic large cell lymphoma. The underlying molecular mechanisms that drive increased inflammation toward textured implants (compared with smooth implants) remain poorly understood. Here, we present the first known case of a patient with Ehlers-Danlos syndrome (EDS) who developed two independent fibrotic capsules around a single textured silicone implant. The patient was found to have one internal capsule tightly adherent to the implant and a second external capsule that was attached to the surrounding tissue. We observed that the internal implant-adherent capsule was composed of a highly aligned and dense collagen network, completely atypical for EDS and indicative of a high mechanical stress environment. In contrast, the external nonadherent capsule, which primarily interacted with the smooth surface of the internal capsule, displayed disorganized collagen fibers with no discernible alignment, classic for EDS. Remarkably, we found that the internal capsule displayed high activation of monocyte chemoattractant protein-1, a mechanoresponsive inflammatory mediator that was not elevated in the disorganized external capsule. Taken together, these findings demonstrate that the tight adhesion between the textured implant surface and the internal capsule creates a high mechanical stress environment, which is responsible for the increased local inflammation observed in the internal capsule. This unique case demonstrates that mechanical stress is able to override genetic defects locally in collagen organization and directly connects the textured surface of implants to prolonged inflammation.

The aim of this study was to further elucidate the molecular mechanisms that mediate pathologic foreign body response (FBR) to biomedical implants. The longevity of biomedical implants is limited by the FBR, which leads to implant failure and patient morbidity. Since the specific molecular mechanisms underlying fibrotic responses to biomedical implants have yet to be fully described, there are currently no targeted approaches to reduce pathologic FBR. We utilized proteomics analysis of human FBR samples to identify potential molecular targets for therapeutic inhibition of FBR. We then employed a murine model of FBR to further evaluate the role of this potential target. We performed histological and immunohistochemical analysis on the murine FBR capsule tissue, as well as single-cell RNA sequencing (scRNA-seq) on cells isolated from the capsules. We identified IQ motif containing GTPase activating protein 1 (IQGAP1) as the most promising of several targets, serving as a central molecular mediator in human and murine FBR compared to control subcutaneous tissue. IQGAP1-deficient mice displayed a significantly reduced FBR compared to wild-type mice as evidenced by lower levels of collagen deposition and maturity. Our scRNA-seq analysis revealed that decreasing IQGAP1 resulted in diminished transcription of mechanotransduction, inflammation, and fibrosis-related genes, which was confirmed on the protein level with immunofluorescent staining. The deficiency of IQGAP1 significantly attenuates FBR by deactivating downstream mechanotransduction signaling, inflammation, and fibrotic pathways. IQGAP1 may be a promising target for rational therapeutic design to mitigate pathologic FBR around biomedical implants.

Burns and other traumatic injuries represent a substantial biomedical burden. The current standard of care for deep injuries is autologous split-thickness skin grafting (STSG), which frequently results in contractures, abnormal pigmentation, and loss of biomechanical function. Currently, there are no effective therapies that can prevent fibrosis and contracture after STSG. Here, we have developed a clinically relevant porcine model of STSG and comprehensively characterized porcine cell populations involved in healing with single-cell resolution. We identified an up-regulation of proinflammatory and mechanotransduction signaling pathways in standard STSGs. Blocking mechanotransduction with a small-molecule focal adhesion kinase (FAK) inhibitor promoted healing, reduced contracture, mitigated scar formation, restored collagen architecture, and ultimately improved graft biomechanical properties. Acute mechanotransduction blockade up-regulated myeloid CXCL10-mediated anti-inflammation with decreased CXCL14-mediated myeloid and fibroblast recruitment. At later time points, mechanical signaling shifted fibroblasts toward profibrotic differentiation fates, and disruption of mechanotransduction modulated mesenchymal fibroblast differentiation states to block those responses, instead driving fibroblasts toward proregenerative, adipogenic states similar to unwounded skin. We then confirmed these two diverging fibroblast transcriptional trajectories in human skin, human scar, and a three-dimensional organotypic model of human skin. Together, pharmacological blockade of mechanotransduction markedly improved large animal healing after STSG by promoting both early, anti-inflammatory and late, regenerative transcriptional programs, resulting in healed tissue similar to unwounded skin. FAK inhibition could therefore supplement the current standard of care for traumatic and burn injuries.

BACKGROUND:The use of biologic mesh to reinforce the abdominal wall in ventral hernia repair has been proposed as a viable alternative to synthetic mesh, particularly for high-risk patients and in contaminated settings. However, a comparison of clinical outcomes between the currently available biologic mesh types has yet to be performed. METHODS:We performed a retrospective analysis of 141 patients who had undergone ventral hernia repair with biologic mesh, including noncross-linked porcine ADM (NC-PADM) (n = 51), cross-linked porcine ADM (C-PADM) (n = 17), reinforced biologic ovine rumen (RBOR) (n = 36), and bovine ADM (BADM) (n = 37) at the Stanford University Medical Center between 2002 and 2020. Postoperative donor site complications and rates of hernia recurrence were compared between patients with different biologic mesh types. RESULTS:= 0.0773) compared with those who had received RBOR. Furthermore, relative risk for hernia recurrence was also higher in all other mesh types compared with RBOR. CONCLUSION/CONCLUSIONS:Our data indicate that RBOR decreases abdominal complications and recurrence rates after ventral hernia repair compared with NC-PADM, C-PADM, and BADM.

Hand injuries often result in significant functional impairments and are rarely completely restored. The spontaneous regeneration of injured appendages, which occurs in salamanders and newts, for example, has been reported in human fingertips after distal amputation, but this type of regeneration is rare in mammals and is incompletely understood. Here, we study fingertip regeneration by amputating murine digit tips, either distally to initiate regeneration, or proximally, causing fibrosis. Using an unbiased microarray analysis, we found that digit tip regeneration is significantly associated with hair follicle differentiation, Wnt, and sonic hedgehog (SHH) signaling pathways. Viral over-expression and genetic knockouts showed the functional significance of these pathways during regeneration. Using transgenic reporter mice, we demonstrated that, while both canonical Wnt and HH signaling were limited to epidermal tissues, downstream hedgehog signaling (through Gli) occurred in mesenchymal tissues. These findings reveal a mechanism for epidermal/mesenchyme interactions, governed by canonical hedgehog signaling, during digit regeneration. Further research into these pathways could lead to improved therapeutic outcomes after hand injuries in humans.

BACKGROUND:Reinforcement of the abdominal wall with synthetic mesh in autologous breast reconstruction using abdominal free tissue transfer decreases the risk of bulging and herniation. However, the impact of the plane of mesh placement on donor site complications has not yet been investigated. METHODS:We performed a retrospective analysis of 312 patients who had undergone autologous breast reconstruction with muscle-sparing transverse rectus abdominis myocutaneous (MS-TRAM) flaps or deep inferior epigastric perforator (DIEP) flaps as well as polypropylene mesh implantation at the donor site. Donor site complications were compared among patients with different flap types and different mesh positions including overlay (n = 90), inlay and overlay (I-O; n = 134), and sublay (n = 88). RESULTS:Abdominal hernias occurred in 2.86% of patients who had undergone MS-TRAM reconstructions and in 2.63% of patients who had undergone DIEP reconstructions. When comparing patients with different mesh positions, donor site complications occurred in 14.4% of patients with overlay mesh, 13.4% of patients with I-O mesh, and 10.2% of patients with sublay mesh (P = 0.68). Abdominal hernias occurred in 4.44% of patients with overlay mesh, 2.24% of patients with I-O mesh, and 2.27% of patients with sublay mesh (P = 0.69). Multivariable logistic regression analysis did not identify a significant association between mesh position and hernia rates as well as wound complications. CONCLUSIONS:Our data indicate that the plane of synthetic mesh placement in relation to the rectus abdominis muscle does not impact the rate of postoperative donor site complications in patients undergoing breast reconstruction with MS-TRAM or DIEP flaps.

Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available. The relationship between the immune response to foreign grafts and their impact on wound healing has not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSGs) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to a fibrotic collagen deposition. We show that macrophage differentiation in response to hSTSG implantation is driven toward regenerative Trem2⁺ subpopulations and found that hydrogel delivery of these cells significantly accelerated wound closure. Our study identifies the preclinical therapeutic potential of Trem2⁺ macrophages to mitigate fibrosis and promote wound healing, providing a novel effective strategy to develop advanced cell therapies for complex wounds.

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.