Longitudinal Assessment of Neurodevelopment in Patients With Nonsyndromic Single-Suture Craniosynostosis: A Retrospective Review of 66 Patients.

The neurodevelopmental consequences of nonsyndromic single-suture (NSS) craniosynostosis are the subject of continued debate. Although the predictive validity of the Bayley Scales of Infant and Toddler Development (Third Edition) (BSID-III) have been questioned, this neurodevelopmental testing battery continues to be widely utilized among multidisciplinary craniofacial teams. The purpose of this study is to evaluate the neurodevelopmental functioning of patients with NSS craniosynostosis before and after surgical correction and the impact of surgical correction on neurodevelopmental trajectory based on BSID-III testing. All patients with NSS craniosynostosis who underwent cranial vault remodeling between 2009 and 2020 were considered for inclusion. Patients who failed to complete BSID-III testing within 2 months of surgery preoperatively and 2 years of surgery postoperatively were excluded. A total of 66 patients met criteria for the study. On language testing, both the preoperative mean score ( P =0.007) and postoperative mean score ( P =0.003) were significantly lower than the population norm. Furthermore, on motor testing, both the preoperative mean score ( P =0.005) and postoperative mean score ( P =0.001) were significantly lower than the population norm. Bayley Scales of Infant and Toddler Development (Third Edition) testing revealed no significant change between preoperative and postoperative neurodevelopmental functioning. Overall, this study suggests that patients with NSS craniosynostosis experience modest delays in language and motor development, which are present before and after cranial vault remodeling. In addition, this study provides evidence that cranial vault remodeling does not significantly impact the neurodevelopmental trajectory. Multicenter st udies and refined neurodevelopmental testing methods are necessary to definitively establish the neurodevelopmental implications of NSS craniosynostosis.

Quantitative perfusion assessment of gastric conduit with indocyanine green dye to predict anastomotic leak after esophagectomy.

Impaired gastric conduit perfusion is a risk factor for anastomotic leak after esophagectomy. The aim of this study is to evaluate the feasibility of intraoperative quantitative assessment of gastric conduit perfusion with indocyanine green fluorescence angiography as a predictor for cervical esophagogastric anastomotic leak after esophagectomy. Indocyanine green fluorescence angiography using the SPY Elite system was performed in patients undergoing a transhiatal or McKeown esophagectomy from July 2015 through December 2020. Ingress (dye uptake) and Egress (dye exit) at two anatomic landmarks (the tip of a conduit and 5 cm from the tip) were assessed. The collected data in the leak group and no leak group were compared by univariate and multivariable analyses. Of 304 patients who were evaluated, 70 patients developed anastomotic leak (23.0%). There was no significant difference in patients' demographic between the groups. Ingress Index, which represents a proportion of blood inflow, at both the tip and 5 cm of the conduit was significantly lower in the leak group (17.9 vs. 25.4% [P = 0.011] and 35.9 vs. 44.6% [P = 0.019], respectively). Ingress Time, which represents an estimated time of blood inflow, at 5 cm of the conduit was significantly higher in the leak group (69.9 vs. 57.1 seconds, P = 0.006). Multivariable analysis suggested that these three variables can be used to predict future leak. Variables of gastric conduit perfusion correlated with the incidence of cervical esophagogastric anastomotic leak. Intraoperative measurement of gastric conduit perfusion can be predictive for anastomotic leak following esophagectomy.

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.

Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification.

Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

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).

Low Prevalence of Clinically Significant Hyponatremia following Cranial Vault Reconstruction for Single-Suture Craniosynostosis.

BACKGROUND: Patients are commonly monitored for hyponatremia after intracranial procedures, yet the prevalence of hyponatremia after cranial vault reconstruction (CVR) remains unclear. The purpose of this study is to define the prevalence, risk factors, and complications of hyponatremia after CVR to optimize postoperative sodium surveillance protocols. METHODS: Patients with nonsyndromic, single-suture craniosynostosis who underwent primary CVR between 2009 and 2020 at Michigan Medicine were included (n = 231). Demographic, intraoperative, and postoperative characteristics were compared by postoperative hyponatremia status at P < 0.05 significance. Hyponatremia was defined as mild (<135 mEq/L), moderate (<130 mEq/L), or severe (<125 mEq/L) based on the lowest postoperative laboratory draw. RESULTS: Twenty-three patients (10.0%) developed mild postoperative hyponatremia. No patient developed moderate or severe postoperative hyponatremia. On multivariable regression, decreased preoperative sodium level (P = 0.03) and decreased preoperative weight (P = 0.02) were significantly associated with mild postoperative hyponatremia. No patient developed complications or required hospital readmission because of hyponatremia. CONCLUSIONS: This large retrospective cohort study of patients with nonsyndromic single-suture craniosynostosis demonstrated a 10% prevalence of mild, clinically inconsequential hyponatremia and 0% prevalence of moderate or severe, clinically significant hyponatremia after primary CVR. Patients with low preoperative sodium level or weight were at increased risk for developing mild postoperative hyponatremia. The results suggest that patients with preoperative sodium greater than 140 mEq/L or preoperative weight greater than 10 kg may be candidates for limited postoperative sodium surveillance; however, future prospective studies are warranted before implementation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Surgical Timing and Neurocognitive Development among Patients with Craniosynostosis: Analysis of Confounders.

BACKGROUND: In 1988, Renier and Marchac asserted that children with craniosynostosis who undergo cranial vault remodeling (CVR) after 12 months of age experience delayed neurocognitive development compared to children who undergo CVR before 12 months of age. The purpose of this study was to identify factors potentially confounding this cause-and-effect relationship. The authors hypothesize that children with socioeconomic disadvantages or comorbid conditions are more likely to undergo CVR after 12 months and may represent a selection bias toward delayed neurocognitive development. METHODS: Patients with nonsyndromic single-suture craniosynostosis who underwent CVR between 2009 and 2020 at Michigan Medicine were included ( n = 227). Sociodemographic and clinical variables were documented. The sample was dichotomized to compare patients who underwent CVR before (early) and after (late) 12 months of age. Statistical analysis was performed at P < 0.05 significance. RESULTS: The early and late groups contained 157 patients and 70 patients, respectively. Compared to the early group, the late group contained a larger proportion of patients who identified as non-White ( P = 0.03), qualified for need-based financial assistance ( P = 0.03), were born preterm ( P < 0.01), or had a comorbid condition ( P < 0.01). Based on preoperative testing, the late group contained a larger proportion of patients with baseline cognitive ( P < 0.001) and language ( P = 0.008) delays relative to the early group. CONCLUSIONS: This study demonstrates that socioeconomic disadvantages and comorbid conditions are prevalent among patients who undergo delayed CVR and may represent a selection bias toward delayed neurocognitive development. Future studies evaluating the relationship between surgical timing and neurocognitive development must control for these factors. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Prescribed Opioid Dosages, Payer Type, and Self-Reported Outcomes After Surgical Procedures in Michigan, 2018-2020.

Importance: Collaborative quality improvement (CQI) models, often supported by private payers, create hospital networks to improve health care delivery. Recently, these systems have focused on opioid stewardship; however, it is unclear whether reduction in postoperative opioid prescribing occurs uniformly across health insurance payer types. Objective: To evaluate the association between insurance payer type, postoperative opioid prescription size, and patient-reported outcomes in a large statewide CQI model. Design, Setting, and Participants: This retrospective cohort study used data from 70 hospitals within the Michigan Surgical Quality Collaborative clinical registry for adult patients (age ≥18 years) undergoing general, colorectal, vascular, or gynecologic surgical procedures between January 1, 2018, and December 31, 2020. Exposure: Insurance type, classified as private, Medicare, or Medicaid. Main Outcomes and Measures: The primary outcome was postoperative opioid prescription size in milligrams of oral morphine equivalents (OME). Secondary outcomes were patient-reported opioid consumption, refill rate, satisfaction, pain, quality of life, and regret about undergoing surgery. Results: A total of 40 149 patients (22 921 [57.1%] female; mean [SD] age, 53 [17] years) underwent surgery during the study period. Within this cohort, 23 097 patients (57.5%) had private insurance, 10 667 (26.6%) had Medicare, and 6385 (15.9%) had Medicaid. Unadjusted opioid prescription size decreased for all 3 groups during the study period from 115 to 61 OME for private insurance patients, from 96 to 53 OME for Medicare patients, and from 132 to 65 OME for Medicaid patients. A total of 22 665 patients received a postoperative opioid prescription and had follow-up data for opioid consumption and refill. The rate of opioid consumption was highest among Medicaid patients throughout the study period (16.82 OME [95% CI, 12.57-21.07 OME] greater than among patients with private insurance) but increased the least over time. The odds of refill significantly decreased over time for patients with Medicaid compared with patients with private insurance (odds ratio, 0.93; 95% CI, 0.89-0.98). Adjusted refill rates for private insurance remained between 3.0% and 3.1% over the study period; adjusted refill rates among Medicare and Medicaid patients decreased from 4.7% to 3.1% and 6.5% to 3.4%, respectively, by the end of the study period. Conclusions and Relevance: In this retrospective cohort study of surgical patients in Michigan from 2018 to 2020, postoperative opioid prescription size decreased across all payer types, and differences between groups narrowed over time. Although funded by private payers, the CQI model appeared to have benefitted patients with Medicare and Medicaid as well.

High-frequency spectral ultrasound imaging (SUSI) visualizes early post-traumatic heterotopic ossification (HO) in a mouse model.

PURPOSE: Early treatment of heterotopic ossification (HO) is currently limited by delayed diagnosis due to limited visualization at early time points. In this study, we validate the use of spectral ultrasound imaging (SUSI) in an animal model to detect HO as early as one week after burn tenotomy. METHODS: Concurrent SUSI, micro CT, and histology at 1, 2, 4, and 9weeks post-injury were used to follow the progression of HO after an Achilles tenotomy and 30% total body surface area burn (n=3-5 limbs per time point). To compare the use of SUSI in different types of injury models, mice (n=5 per group) underwent either burn/tenotomy or skin incision injury and were imaged using a 55MHz probe on VisualSonics VEVO 770 system at one week post injury to evaluate the ability of SUSI to distinguish between edema and HO. Average acoustic concentration (AAC) and average scatterer diameter (ASD) were calculated for each ultrasound image frame. Micro CT was used to calculate the total volume of HO. Histology was used to confirm bone formation. RESULTS: Using SUSI, HO was visualized as early as 1week after injury. HO was visualized earliest by 4weeks after injury by micro CT. The average acoustic concentration of HO was 33% more than that of the control limb (n=5). Spectroscopic foci of HO present at 1week that persisted throughout all time points correlated with the HO present at 9weeks on micro CT imaging. CONCLUSION: SUSI visualizes HO as early as one week after injury in an animal model. SUSI represents a new imaging modality with promise for early diagnosis of HO.

Local and Circulating Endothelial Cells Undergo Endothelial to Mesenchymal Transition (EndMT) in Response to Musculoskeletal Injury.

Endothelial-to-mesenchymal transition (EndMT) has been implicated in a variety of aberrant wound healing conditions. However, unambiguous evidence of EndMT has been elusive due to limitations of in vitro experimental designs and animal models. In vitro experiments cannot account for the myriad ligands and cells which regulate differentiation, and in vivo tissue injury models may induce lineage-independent endothelial marker expression in mesenchymal cells. By using an inducible Cre model to mark mesenchymal cells (Scx-creERT/tdTomato + ) prior to injury, we demonstrate that musculoskeletal injury induces expression of CD31, VeCadherin, or Tie2 in mesenchymal cells. VeCadherin and Tie2 were expressed in non-endothelial cells (CD31-) present in marrow from uninjured adult mice, thereby limiting the specificity of these markers in inducible models (e.g. VeCadherin- or Tie2-creERT). However, cell transplantation assays confirmed that endothelial cells (ΔVeCadherin/CD31+/CD45-) isolated from uninjured hindlimb muscle tissue undergo in vivo EndMT when transplanted directly into the wound without intervening cell culture using PDGFRα, Osterix (OSX), SOX9, and Aggrecan (ACAN) as mesenchymal markers. These in vivo findings support EndMT in the presence of myriad ligands and cell types, using cell transplantation assays which can be applied for other pathologies implicated in EndMT including tissue fibrosis and atherosclerosis. Additionally, endothelial cell recruitment and trafficking are potential therapeutic targets to prevent EndMT.

mTOR inhibition and BMP signaling act synergistically to reduce muscle fibrosis and improve myofiber regeneration.

Muscle trauma is highly morbid due to intramuscular scarring, or fibrosis, and muscle atrophy. Studies have shown that bone morphogenetic proteins (BMPs) reduce muscle atrophy. However, increased BMP signaling at muscle injury sites causes heterotopic ossification, as seen in patients with fibrodysplasia ossificans progressiva (FOP), or patients with surgically placed BMP implants for bone healing. We use a genetic mouse model of hyperactive BMP signaling to show the development of intramuscular fibrosis surrounding areas of ectopic bone following muscle injury. Rapamycin, which we have previously shown to eliminate ectopic ossification in this model, also eliminates fibrosis without reducing osteogenic differentiation, suggesting clinical value for patients with FOP and with BMP implants. Finally, we use reporter mice to show that BMP signaling is positively associated with myofiber cross-sectional area. These findings underscore an approach in which 2 therapeutics (rapamycin and BMP ligand) can offset each other, leading to an improved outcome.

Characterization of Heterotopic Ossification Using Radiographic Imaging: Evidence for a Paradigm Shift.

Heterotopic ossification (HO) is the growth of extra-skeletal bone which occurs following trauma, burns, and in patients with genetic bone morphogenetic protein (BMP) receptor mutations. The clinical and laboratory evaluation of HO is dependent on radiographic imaging to identify and characterize these lesions. Here we show that despite its inadequacies, plain film radiography and single modality microCT continue to serve as a primary method of HO imaging in nearly 30% of published in vivo literature. Furthermore, we demonstrate that detailed microCT analysis is superior to plain film and single modality microCT radiography specifically in the evaluation of HO formed through three representative models due to its ability to 1) define structural relationships between growing extra-skeletal bone and normal, anatomic bone, 2) provide accurate quantification and growth rate based on volume of the space-occupying lesion, thereby facilitating assessments of therapeutic intervention, 3) identify HO at earlier times allowing for evaluation of early intervention, and 4) characterization of metrics of bone physiology including porosity, tissue mineral density, and cortical and trabecular volume. Examination of our trauma model using microCT demonstrated two separate areas of HO based on anatomic location and relationship with surrounding, normal bone structures. Additionally, microCT allows HO growth rate to be evaluated to characterize HO progression. Taken together, these data demonstrate the need for a paradigm shift in the evaluation of HO towards microCT as a standard tool for imaging.