Clinical, mechanistic, and therapeutic landscape of cutaneous fibrosis.

When dysregulated, skin fibrosis can lead to a multitude of pathologies. We provide a framework for understanding the wide clinical spectrum, mechanisms, and management of cutaneous fibrosis encompassing a variety of matrix disorders, fibrohistiocytic neoplasms, injury-induced scarring, and autoimmune scleroses. Underlying such entities are common mechanistic pathways that leverage morphogenic signaling, immune activation, and mechanotransduction to modulate fibroblast function. In light of the limited array of available treatments for cutaneous fibrosis, scientific insights have opened new therapeutic and investigative avenues for conditions that still lack effective interventions.

Understanding the Foreign Body Response via Single-Cell Meta-Analysis.

Foreign body response (FBR) is a universal reaction to implanted biomaterial that can affect the function and longevity of the implant. A few studies have attempted to identify targets for treating FBR through the use of single-cell RNA sequencing (scRNA-seq), though the generalizability of these findings from an individual study may be limited. In our study, we perform a meta-analysis of scRNA-seq data from all available FBR mouse studies and integrate these data to identify gene signatures specific to FBR across different models and anatomic locations. We identify subclusters of fibroblasts and macrophages that emerge in response to foreign bodies and characterize their signaling pathways, gene ontology terms, and downstream mediators. The fibroblast subpopulations enriched in the setting of FBR demonstrated significant signaling interactions in the transforming growth factor-beta (TGF-ß) signaling pathway, with known pro-fibrotic mediators identified as top expressed genes in these FBR-derived fibroblasts. In contrast, FBR-enriched macrophage subclusters highly expressed pro-fibrotic and pro-inflammatory mediators downstream of tumor necrosis factor (TNF) signaling. Cell-cell interactions were additionally interrogated using CellChat, with identification of key signaling interactions enriched between fibroblasts and macrophages in FBR. By combining multiple FBR datasets, our meta-analysis study identifies common cell-specific gene signatures enriched in foreign body reactions, providing potential therapeutic targets for patients requiring medical implants across a myriad of devices and indications.

Role of ferroptosis in radiation-induced soft tissue injury.

Ionizing radiation has been pivotal in cancer therapy since its discovery. Despite its therapeutic benefits, IR causes significant acute and chronic complications due to DNA damage and the generation of reactive oxygen species, which harm nucleic acids, lipids, and proteins. While cancer cells are more vulnerable to ionizing radiation due to their inefficiency in repairing damage, healthy cells in the irradiated area also suffer. Various types of cell death occur, including apoptosis, necrosis, pyroptosis, autophagy-dependent cell death, immunogenic cell death, and ferroptosis. Ferroptosis, driven by iron-dependent lipid peroxide accumulation, has been recognized as crucial in radiation therapy's therapeutic effects and complications, with extensive research across various tissues. This review aims to summarize the pathways involved in radiation-related ferroptosis, findings in different organs, and drugs targeting ferroptosis to mitigate its harmful effects.

Single-cell transcriptional analysis of irradiated skin reveals changes in fibroblast subpopulations and variability in caveolin expression.

BACKGROUND: Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury. METHODS: Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing. RESULTS: Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0-4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses. CONCLUSION: In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.

Hematoxylin and Eosin Architecture Uncovers Clinically Divergent Niches in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the only cancers with an increasing incidence rate and is often associated with intra- and peri-tumoral scarring, referred to as desmoplasia. This scarring is highly heterogeneous in extracellular matrix (ECM) architecture and plays complex roles in both tumor biology and clinical outcomes that are not yet fully understood. Using hematoxylin and eosin (H&E), a routine histological stain utilized in existing clinical workflows, we quantified ECM architecture in 85 patient samples to assess relationships between desmoplastic architecture and clinical outcomes such as survival time and disease recurrence. By utilizing unsupervised machine learning to summarize a latent space across 147 local (e.g., fiber length, solidity) and global (e.g., fiber branching, porosity) H&E-based features, we identified a continuum of histological architectures that were associated with differences in both survival and recurrence. Furthermore, we mapped H&E architectures to a CO-Detection by indEXing (CODEX) reference atlas, revealing localized cell- and protein-based niches associated with outcome-positive versus outcome-negative scarring in the tumor microenvironment. Overall, our study utilizes standard H&E staining to uncover clinically relevant associations between desmoplastic organization and PDAC outcomes, offering a translatable pipeline to support prognostic decision-making and a blueprint of spatial-biological factors for modeling by tissue engineering methods.

Ferroptosis Inhibition with Deferoxamine Alleviates Radiation-Induced Fibrosis.

Background: Radiation-induced fibrosis (RIF) is a debilitating sequelae of radiation therapy that has been shown to improve with topical treatment with the iron chelator deferoxamine (DFO). We investigated whether DFO exerts this effect through attenuation of ferroptosis, a recently described iron-dependent pathway of cell death. Methods: Adult C57BL/6J mice were treated with topical DFO or ferrostastin-1 (Fer-1) and irradiated with 30 Grays of ionizing radiation to the dorsal skin to promote development of chronic RIF. Immunofluorescent staining with 4-hydroxynonenal (4-HNE) antibody was carried out directly following irradiation to assess ferroptosis activity. Perfusion testing with laser Doppler was performed throughout the healing interval. Eight weeks following radiation, dorsal skin was harvested and analyzed histologically and biomechanically. Results: Immunohistochemical staining demonstrated lower presence of 4-HNE in non-irradiated skin, DFO-treated skin, and Fer-1-treated skin compared to irradiated, untreated skin. DFO resulted in histological measurements (dermal thickness and collagen content) that resembled normal skin, while Fer-1 treatment yielded less significant improvements. These results were mirrored by analysis of extracellular matrix ultrastructure and biomechanical testing, which recapitulated the ability of topical DFO treatment to alleviate RIF across these parameters while Fer-1 resulted in less notable improvement. Finally, perfusion levels in DFO treated irradiated skin were similar to measurements in normal skin, while Fer-1 treatment did not impact this feature. Conclusions: Ferroptosis contributes to the development of RIF and attenuation of this process leads to reduced skin injury. DFO further improves RIF through additional enhancement of perfusion not seen with Fer-1.

Deferoxamine topical cream superior to patch in rescuing radiation-induced fibrosis of unwounded and wounded skin.

Topical patch delivery of deferoxamine (DFO) has been studied as a treatment for this fibrotic transformation in irradiated tissue. Efficacy of a novel cream formulation of DFO was studied as a RIF therapeutic in unwounded and excisionally wounded irradiated skin. C57BL/6J mice underwent 30 Gy of radiation to the dorsum followed by 4 weeks of recovery. In a first experiment, mice were separated into six conditions: DFO 50 mg cream (D50), DFO 100 mg cream (D100), soluble DFO injections (DI), DFO 1 mg patch (DP), control cream (Vehicle), and irradiated untreated skin (IR). In a second experiment, excisional wounds were created on the irradiated dorsum of mice and then divided into four treatment groups: DFO 100 mg Cream (W-D100), DFO 1 mg patch (W-DP), control cream (W-Vehicle), and irradiated untreated wounds (W-IR). Laser Doppler perfusion scans, biomechanical testing, and histological analysis were performed. In irradiated skin, D100 improved perfusion compared to D50 or DP. Both D100 and DP enhanced dermal characteristics, including thickness, collagen density and 8-isoprostane staining compared to untreated irradiated skin. D100 outperformed DP in CD31 staining, indicating higher vascular density. Extracellular matrix features of D100 and DP resembled normal skin more closely than DI or control. In radiated excisional wounds, D100 facilitated faster wound healing and increased perfusion compared to DP. The 100 mg DFO cream formulation rescued RIF of unwounded irradiated skin and improved excisional wound healing in murine skin relative to patch delivery of DFO.

A Review of Radiation-Induced Vascular Injury and Clinical Impact.

ABSTRACT: The number of cancer survivors continues to increase because of advances in therapeutic modalities. Along with surgery and chemotherapy, radiotherapy is a commonly used treatment modality in roughly half of all cancer patients. It is particularly helpful in the oncologic treatment of patients with breast, head and neck, and prostate malignancies. Unfortunately, among patients receiving radiation therapy, long-term sequalae are often unavoidable, and there is accumulating clinical evidence suggesting significant radiation-related damage to the vascular endothelium. Ionizing radiation has been known to cause obliterative fibrosis and increased wall thickness in irradiated blood vessels. Clinically, these vascular changes induced by ionizing radiation can pose unique surgical challenges when operating in radiated fields. Here, we review the relevant literature on radiation-induced vascular damage focusing on mechanisms and signaling pathways involved and highlight microsurgical anastomotic outcomes after radiotherapy. In addition, we briefly comment on potential therapeutic strategies, which may have the ability to mitigate radiation injury to the vascular endothelium.

Establishing a Xenograft Model with CD-1 Nude Mice to Study Human Skin Wound Repair.

BACKGROUND: A significant gap exists in the translatability of small-animal models to human subjects. One important factor is poor laboratory models involving human tissue. Thus, the authors have created a viable postnatal human skin xenograft model using athymic mice. METHODS: Discarded human foreskins were collected following circumcision. All subcutaneous tissue was removed from these samples sterilely. Host CD-1 nude mice were then anesthetized, and dorsal skin was sterilized. A 1.2-cm-diameter, full-thickness section of dorsal skin was excised. The foreskin sample was then placed into the full-thickness defect in the host mice and sutured into place. Xenografts underwent dermal wounding using a 4-mm punch biopsy after engraftment. Xenografts were monitored for 14 days after wounding and then harvested. RESULTS: At 14 days postoperatively, all mice survived the procedure. Grossly, the xenograft wounds showed formation of a human scar at postoperative day 14. Hematoxylin and eosin and Masson trichome staining confirmed scar formation in the wounded human skin. Using a novel artificial intelligence algorithm using picrosirius red staining, scar formation was confirmed in human wounded skin compared with the unwounded skin. Histologically, CD31 + immunostaining confirmed vascularization of the xenograft. The xenograft exclusively showed human collagen type I, CD26 + , and human nuclear antigen in the human scar without any staining of these human markers in the murine skin. CONCLUSION: The proposed model demonstrates wound healing to be a local response from tissue resident human fibroblasts and allows for reproducible evaluation of human skin wound repair in a preclinical model. CLINICAL RELEVANCE STATEMENT: Radiation-induced fibrosis is a widely prevalent clinical phenomenon without a well-defined treatment at this time. This study will help establish a small-animal model to better understand and develop novel therapeutics to treat irradiated human skin.

Desmoplastic stromal signatures predict patient outcomes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related death. Hallmarks include desmoplasia with variable extracellular matrix (ECM) architecture and a complex microenvironment with spatially defined tumor, stromal, and immune populations. Nevertheless, the role of desmoplastic spatial organization in patient/tumor variability remains underexplored, which we elucidate using two technologies. First, we quantify ECM patterning in 437 patients, revealing architectures associated with disease-free and overall survival. Second, we spatially profile the cellular milieu of 78 specimens using codetection by indexing, identifying an axis of pro-inflammatory cell interactions predictive of poorer outcomes. We discover that clinical characteristics, including neoadjuvant chemotherapy status, tumor stage, and ECM architecture, correlate with differential stromal-immune organization, including fibroblast subtypes with distinct niches. Lastly, we define unified signatures that predict survival with areas under the receiver operating characteristic curve (AUCs) of 0.872-0.903, differentiating survivorship by 655 days. Overall, our findings establish matrix ultrastructural and cellular organizations of fibrosis linked to poorer outcomes.

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing.

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.

Understanding the Role of Adipocytes and Fibroblasts in Cancer.

ABSTRACT: Cancer is currently the second leading cause of death in the United States. There is increasing evidence that the tumor microenvironment (TME) is pivotal for tumorigenesis and metastasis. Recently, adipocytes and cancer-associated fibroblasts (CAFs) in the TME have been shown to play a major role in tumorigenesis of different cancers, specifically melanoma. Animal studies have shown that CAFs and adipocytes within the TME help tumors evade the immune system, for example, by releasing chemokines to blunt the effectiveness of the host defense. Although studies have identified that adipocytes and CAFs play a role in tumorigenesis, adipocyte transition to fibroblast within the TME is fairly unknown. This review intends to elucidate the potential that adipocytes may have to transition to fibroblasts and, as part of the TME, a critical role that CAFs may play in affecting the growth and invasion of tumor cells. Future studies that illuminate the function of adipocytes and CAFs in the TME may pave way for new antitumor therapies.

Medical Biology of Cancer-Associated Fibroblasts in Pancreatic Cancer.

Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF-tumor cell interaction, as well as CAF-immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

Circulating and extracellular vesicle-derived microRNAs as biomarkers in bone-related diseases.

MicroRNAs (miRNA) are small non-coding RNA molecules that regulate posttranscriptional gene expression by repressing messengerRNA-targets. MiRNAs are abundant in many cell types and are secreted into extracellular fluids, protected from degradation by packaging in extracellular vesicles. These circulating miRNAs are easily accessible, disease-specific and sensitive to small changes, which makes them ideal biomarkers for diagnostic, prognostic, predictive or monitoring purposes. Specific miRNA signatures can be reflective of disease status and development or indicators of poor treatment response. This is especially important in malignant diseases, as the ease of accessibility of circulating miRNAs circumvents the need for invasive tissue biopsy. In osteogenesis, miRNAs can act either osteo-enhancing or osteo-repressing by targeting key transcription factors and signaling pathways. This review highlights the role of circulating and extracellular vesicle-derived miRNAs as biomarkers in bone-related diseases, with a specific focus on osteoporosis and osteosarcoma. To this end, a comprehensive literature search has been performed. The first part of the review discusses the history and biology of miRNAs, followed by a description of different types of biomarkers and an update of the current knowledge of miRNAs as biomarkers in bone related diseases. Finally, limitations of miRNAs biomarker research and future perspectives will be presented.

Reduction of Tendon Fibrosis Using Galectin-3 Inhibitors.

BACKGROUND: Fibrosis is a complication of both tendon injuries and repairs. We aim to develop a mouse model to assess tendon fibrosis and to identify an antifibrotic agent capable of overcoming tendon fibrosis. METHODS: Adult C57Bl/6 mice underwent a skin incision to expose the Achilles tendon, followed by 50% tendon injury and abrasion with sandpaper. Sham surgeries were conducted on contralateral hindlimbs. Histology and immunofluorescent staining for fibrotic markers (Col1, α-SMA) were used to confirm that the model induced tendon fibrosis. A second experiment was conducted to further examine the role of α-SMA in adhesion formation using α-SMA.mTmG mice (6-8 weeks old) (n=3) with the same injury model. The control group (tendon injury) was compared to the sham group, using the contralateral limb with skin incision only. A second experiment was conducted to further examine the role of α-SMA in adhesion formation using α-SMA.mTmG mice (6-8 weeks old) (n=3) with the same injury model. The control group (tendon injury) was compared to the sham group, using the contralateral limb with skin incision only. Lastly, α-SMA.mTmG mice were randomized to either condition 1. Tendon injury (control group) or 2. Tendon injury with Galectin-3 inhibitor (Gal3i) treatment at time of injury (treatment group). RESULTS: Histological analyses confirmed tendon thickening and collagen deposition after tendon injury and abrasion compared to control. Immunofluorescence showed higher levels of Col1 and α-SMA protein expression after injury compared to sham (*p<0.05). RT-qPCR also demonstrated increased gene expression of Col1 and α-SMA after injury compared to sham (*p<0.05). Gal3 protein expression also increased after injury and co-localized with α-SMA positive fibroblasts surrounding the fibrotic tendon. Gal3i treatment decreased collagen deposition and scarring observed in the treatment group (*p<0.05). Flow cytometry analysis further showed reduced numbers of profibrotic fibroblasts (CD26+) in the treatment compared to the control group (*p<0.05). CONCLUSIONS: Our study provides a reproducible and reliable model to investigate tendon fibrosis. Findings suggest the potential of Gal3i to overcome fibrosis resulting from tendon injuries.

The effects of mechanical force on fibroblast behavior in cutaneous injury.

Wound healing results in the formation of scar tissue which can be associated with functional impairment, psychological stress, and significant socioeconomic cost which exceeds 20 billion dollars annually in the United States alone. Pathologic scarring is often associated with exaggerated action of fibroblasts and subsequent excessive accumulation of extracellular matrix proteins which results in fibrotic thickening of the dermis. In skin wounds, fibroblasts transition to myofibroblasts which contract the wound and contribute to remodeling of the extracellular matrix. Mechanical stress on wounds has long been clinically observed to result in increased pathologic scar formation, and studies over the past decade have begun to uncover the cellular mechanisms that underly this phenomenon. In this article, we will review the investigations which have identified proteins involved in mechano-sensing, such as focal adhesion kinase, as well as other important pathway components that relay the transcriptional effects of mechanical forces, such as RhoA/ROCK, the hippo pathway, YAP/TAZ, and Piezo1. Additionally, we will discuss findings in animal models which show the inhibition of these pathways to promote wound healing, reduce contracture, mitigate scar formation, and restore normal extracellular matrix architecture. Recent advances in single cell RNA sequencing and spatial transcriptomics and the resulting ability to further characterize mechanoresponsive fibroblast subpopulations and the genes that define them will be summarized. Given the importance of mechanical signaling in scar formation, several clinical treatments focused on reducing tension on the wound have been developed and are described here. Finally, we will look toward future research which may reveal novel cellular pathways and deepen our understanding of the pathogenesis of pathologic scarring. The past decade of scientific inquiry has drawn many lines connecting these cellular mechanisms that may lead to a map for the development of transitional treatments for patients on the path to scarless healing.

A Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue.

BACKGROUND: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. METHODS: The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions. RESULTS: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. CONCLUSIONS: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

An Inexpensive 3D Printed Mouse Model of Successful, Complication-free Long Bone Distraction Osteogenesis.

Distraction osteogenesis (DO) is used for skeletal defects; however, up to 50% of cases exhibit complications. Previous mouse models of long bone DO have been anecdotally hampered by postoperative complications, expense, and availability. To improve clinical techniques, cost-effective, reliable animal models are needed. Our focus was to develop a new mouse tibial distractor, hypothesized to result in successful, complication-free DO. Methods: A lightweight tibial distractor was developed using CAD and 3D printing. The device was fixed to the tibia of C57Bl/6J mice prior to osteotomy. Postoperatively, mice underwent 5 days latency, 10 days distraction (0.15 mm every 12 hours), and 28 days consolidation. Bone regeneration was examined on postoperative day 43 using micro-computed tomography (µCT) and Movat's modified pentachrome staining on histology (mineralized volume fraction and pixels, respectively). Costs were recorded. We compared cohorts of 11 mice undergoing sham, DO, or acute lengthening (distractor acutely lengthened 3.0 mm). Results: The histological bone regenerate was significantly increased in DO (1,879,257 ± 155,415 pixels) compared to acute lengthening (32847 ± 1589 pixels) (P < 0.0001). The mineralized volume fraction (bone/total tissue volume) of the regenerate was significantly increased in DO (0.9 ± 0.1) compared to acute lengthening (0.7 ± 0.1) (P < 0.001). There was no significant difference in bone regenerate between DO and sham. The distractor was relatively low cost ($11), with no complications. Conclusions: Histology and µCT analysis confirmed that the proposed tibial DO model resulted in successful bone formation. Our model is cost-effective and reproducible, enabling implementation in genetically dissectible transgenic mice.