Use of wound edge inversion (epibole) to generate recalcitrant and inflamed diabetic wounds.

Robust and predictive pre-clinical models of recalcitrant diabetic wounds are critical for advancing research efforts toward improving healing. Murine models have logistic and genetic benefits versus larger animals; however, native murine healing inadequately represents clinically recalcitrant wounds in humans. Furthermore, current humanization techniques employing devices, deleterious mutations or chemical agents each carry model-specific limitations. To better replicate human wounds in a mouse, we developed a novel wound-edge inversion (WEI) technique that mimics the architecture of epibole and mitigates contracture, epithelialization, and consequently wound closure. In this study, we evaluated the reliability and durability of the WEI model in wild-type and obese diabetic mice and compared to healing after (i) punch biopsy, (ii) mechanical/silicone stenting or (iii) exogenous oxidative stressors. In wild-type mice, WEI demonstrated favourable closure characteristics compared to both control and stented wounds, however, wounds progressed to closure by 4 weeks. In contrast, diabetic WEI wounds persisted for 6-10 weeks with reduced contracture and epithelialization. In both diabetic and wild-type mice, WEI sites demonstrated persistence of inflammatory populations, absence of epithelialization, and histologic presence of alpha-SMA positive granulation tissue when compared to controls. We conclude that the WEI technique is particularly valuable for modelling recalcitrant diabetic wounds with sustained inflammation and dysfunctional healing.

Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis.

Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-ß1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-ß1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

Coordinating Tissue Regeneration Through Transforming Growth Factor-ß Activated Kinase 1 Inactivation and Reactivation.

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-ß activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778.

Characterizing the Circulating Cell Populations in Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) occurs secondary to trauma, causing pain and functional limitations. Identification of the cells that contribute to HO is critical to the development of therapies. Given that innate immune cells and mesenchymal stem cells are known contributors to HO, we sought to define the contribution of these populations to HO and to identify what, if any, contribution circulating populations have to HO. A shared circulation was obtained using a parabiosis model, established between an enhanced green fluorescent protein-positive/luciferase+ donor and a same-strain nonreporter recipient mouse. The nonreporter mouse received Achilles tendon transection and dorsal burn injury to induce HO formation. Bioluminescence imaging and immunostaining were performed to define the circulatory contribution of immune and mesenchymal cell populations. Histologic analysis showed circulating cells present throughout each stage of the developing HO anlagen. Circulating cells were present at the injury site during the inflammatory phase and proliferative period, with diminished contribution in mature HO. Immunostaining demonstrated that most early circulatory cells were from the innate immune system; only a small population of mesenchymal cells were present in the HO. We demonstrate the time course of the participation of circulatory cells in trauma-induced HO and identify populations of circulating cells present in different stages of HO. These findings further elucidate the relative contribution of local and systemic cell populations to HO.

Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon.

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomatofl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

Strategic Targeting of Multiple BMP Receptors Prevents Trauma-Induced Heterotopic Ossification.

Trauma-induced heterotopic ossification (tHO) is a condition of pathologic wound healing, defined by the progressive formation of ectopic bone in soft tissue following severe burns or trauma. Because previous studies have shown that genetic variants of HO, such as fibrodysplasia ossificans progressiva (FOP), are caused by hyperactivating mutations of the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1/ALK2, studies evaluating therapies for HO have been directed primarily toward drugs for this specific receptor. However, patients with tHO do not carry known T1-BMPR mutations. Here we show that, although BMP signaling is required for tHO, no single T1-BMPR (ACVR1/ALK2, BMPR1a/ALK3, or BMPR1b/ALK6) alone is necessary for this disease, suggesting that these receptors have functional redundancy in the setting of tHO. By utilizing two different classes of BMP signaling inhibitors, we developed a translational approach to treatment, integrating treatment choice with existing diagnostic options. Our treatment paradigm balances either immediate therapy with reduced risk for adverse effects (Alk3-Fc) or delayed therapy with improved patient selection but greater risk for adverse effects (LDN-212854).

Analysis of Bone-Cartilage-Stromal Progenitor Populations in Trauma Induced and Genetic Models of Heterotopic Ossification.

Heterotopic ossification (HO), the formation of extra-skeletal bone in soft tissues, is a pathologic process occurring after substantial burns or trauma, or in patients with type I bone morphogenetic protein (BMP) receptor hyperactivating mutations. Identifying the cells responsible for de novo bone formation during adulthood is of critical importance for therapeutic and regenerative purposes. Using a model of trauma-induced HO with hind limb Achilles' tenotomy and dorsal burn injury and a genetic nontrauma HO model (Nfatc1-Cre/caAcvr1(fl/wt) ), we demonstrate enrichment of previously defined bone-cartilage-stromal progenitor cells (BCSP: AlphaV+/CD105+/Tie2-/CD45-/Thy1-/6C3-) at the site of HO formation when compared with marrow isolated from the ipsilateral hind limb, or from tissue of the contralateral, uninjured hind limb. Upon transplantation into tenotomy sites soon after injury, BCSPs isolated from neonatal mice or developing HO incorporate into the developing lesion in cartilage and bone and express chondrogenic and osteogenic transcription factors. Additionally, BCSPs isolated from developing HO similarly incorporate into new HO lesions upon transplantation. Finally, adventitial cells, but not pericytes, appear to play a supportive role in HO formation. Our findings indicate that BCSPs contribute to de novo bone formation during adulthood and may hold substantial regenerative potential. Stem Cells 2016;34:1692-1701.

Combined reflectance and Raman spectroscopy to assess degree of in vivo angiogenesis after tissue injury.

BACKGROUND: Angiogenesis, the formation of blood vessels, is a critical aspect of wound healing. Disorders of wound healing are often characterized by lack of angiogenesis, a condition frequently observed in aging and diabetic patients. Current techniques for assessing blood at injury sites are limited to contrast-imaging, including angiography. However, these techniques do not directly observe oxygenation of blood and are not amenable to serial evaluation. A multimodal noninvasive reflectance and Raman spectrometer have been proposed to help clinicians as a point-of-care tool to interrogate local angiogenesis and tissue architecture, respectively. The spectrometer system is a rapid, noninvasive, and label-free technology well-suited for the clinical environment. MATERIALS AND METHODS: To demonstrate feasibility, the spectrometer system was used to interrogate angiogenesis serially over 9 wk as a result of heterotopic ossification (HO) development in a validated murine model. End-stage HO was confirmed by micro-computed tomography. RESULTS: Our preliminary results suggest that reflectance spectroscopy can be used to delineate vessel formation and that pathologic wounds may be characterized by unique spectra. In our model, HO formed at sites 1-3, whereas sites 4 and 5 did not have radiographic evidence of HO. CONCLUSIONS: A point-of-care system like that demonstrated here shows potential as a noninvasive tool to assess local angiogenesis and tissue architecture that may allow for timely intervention in a clinical setting.

BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints.

BMP signaling mediated by ACVR1 plays a critical role for development of multiple structures including the cardiovascular and skeletal systems. While deficient ACVR1 signaling impairs normal embryonic development, hyperactive ACVR1 function (R206H in humans and Q207D mutation in mice, ca-ACVR1) results in formation of heterotopic ossification (HO). We developed a mouse line, which conditionally expresses ca-ACVR1 with Nfatc1-Cre(+) transgene. Mutant mice developed ectopic cartilage and bone at the distal joints of the extremities including the interphalangeal joints and hind limb ankles as early as P4 in the absence of trauma or exogenous bone morphogenetic protein (BMP) administration. Micro-CT showed that even at later time points (up to P40), cartilage and bone development persisted at the affected joints most prominently in the ankle. Interestingly, this phenotype was not present in areas of bone outside of the joints - tibia are normal in mutants and littermate controls away from the ankle. These findings demonstrate that this model may allow for further studies of heterotopic ossification, which does not require the use of stem cells, direct trauma or activation with exogenous Cre gene administration.

Vitamin D3 Improves Adipose Stromal Cell Survival and Human Fat Graft Retention in Xenograft Model.

Adipose stem cells are considered one of the primary drivers of autologous fat graft biologic activity and survival. We have previously demonstrated that hormonally active VD3 improved adipose stem cell viability in ex vivo and in vivo fat grafting models. In this study, we evaluated the inactive form of VD3 (cholecalciferol) on adipose stromal cell (ASC) phenotype during hypoxia and the subsequent effect on human fat graft retention in the xenograft model. Lipoaspirate collected from six human donors was used for ex-vivo particle culture studies and isolated ASC studies. Adipose particles were treated with increasing doses of VD3 to determine impact on ASC survival. Expanded stromal cells were treated with VD3 during hypoxic culture and assessed for viability, apoptosis, mitochondrial activity, and nitric oxide release via caspase, DAF-FM, or TMRM. Finally, forty Nu/J mice receiving bilateral dorsal human lipoaspirate were treated thrice weekly with 1) Vehicle control, 2) 50ng calcitriol, 3) 50ng VD3, 4) 500ng VD3, and 5) 5000ng VD3 for 12 weeks, n=8 per group. Graft weight, volume, and architecture were analyzed. Adipose particles treated with dose escalating VD3 had significantly increased ASC viability compared to control (p<0.01). Under hypoxia, ASCs treated with 1nM VD3 had significantly greater viability than untreated and pre-treated cells (p<0.01, p<0.01) and significantly lower apoptosis-to-viability ratio (p<0.01). ASCs pre-treated with 1nM VD3 had significantly lower nitric oxide release (p<0.05) and lower mitochondrial polarization (p<0.05) compared to controls. In vivo results showed mice receiving 5000ng VD3 had significantly greater graft weight (p<0.05) and volume (p<0.05) after 12 weeks of treatment compared to controls. Grafts had enhanced neovascularization, intact adipocyte architecture, and absence of oil cysts. VD3 is an over-the-counter nutritional supplement with a known safety profile in humans. Our xenograft model suggests administering VD3 at the time of surgery may significantly improve fat graft retention.

Evolution of the Push-2-Spin Fat Graft Processing Device: Enhancing Efficiency and Reducing Risk of Contamination.

Background: Small-volume fat graft efficiency is a critical determinant of the cost and material effectiveness of aesthetic fat grafting in the clinical space. Recent development of devices, such as the Push-2-Spin (P2S) system (Pittsburgh, PA), has improved upon the process by yielding a rapid, handheld, multi-use system to minimize operative time and mess. Objectives: In this study, the authors describe further technical innovations on the P2S prototype that improve operative ease of use, time, and safety. Methods: Abdominoplasty samples were obtained as discarded tissue. Lipoaspirate was collected utilizing a 3.0 mm liposuction cannula and processed through centrifugation (Coleman technique), gauze (telfa) rolling, mesh straining, the tabletop P2S device (prototype), or the P2S handheld (P2S-H) device. Operative processing time, spin time, oil fraction, stromal vascular fraction (SVF) yield and viability, and adipocyte viability were assessed to compare the efficacy and viability of each device/technique. Blood agar smears of lipoaspirate were performed to assess for risk of contamination. Results: The P2S-H device outperformed its prior iteration in rotary and processing speed and was significantly faster than each other technique assessed. Furthermore, the use of an inline system offered significant advantages over open-air techniques in terms of resistance to contamination. Serial use characteristics were assessed; under these conditions, oil yield as well as adipocyte and SVF number and viability was similar between all techniques. Conclusions: The technical advancements to the P2S system which enable single-unit, handheld operation significantly improve operative time and minimize space requirements. This operative quality of life improvement comes at no cost to the efficacy of oil extraction, cellular yield, or cell viability.

Disruption of Neutrophil Extracellular Traps (NETs) Links Mechanical Strain to Post-traumatic Inflammation.

Inflammation after trauma is both critical to normal wound healing and may be highly detrimental when prolonged or unchecked with the potential to impair physiologic healing and promote de novo pathology. Mechanical strain after trauma is associated with impaired wound healing and increased inflammation. The exact mechanisms behind this are not fully elucidated. Neutrophil extracellular traps (NETs), a component of the neutrophil response to trauma, are implicated in a range of pro-inflammatory conditions. In the current study, we evaluated their role in linking movement and inflammation. We found that a link exists between the disruption and amplification of NETs which harbors the potential to regulate the wound's response to mechanical strain, while leaving the initial inflammatory signal necessary for physiologic wound healing intact.

Evaluation of Salivary Cytokines for Diagnosis of both Trauma-Induced and Genetic Heterotopic Ossification.

PURPOSE: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. METHODS: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt ). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. RESULTS: Levels of TNF-α and IL-1ß peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1ß, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). CONCLUSION: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.

Direct Mouse Trauma/Burn Model of Heterotopic Ossification.

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.