ECM-Focused Proteomic Analysis of Ear Punch Regeneration in Acomys Cahirinus.

In mammals, significant injury is generally followed by the formation of a fibrotic scar which provides structural integrity but fails to functionally restore damaged tissue. Spiny mice of the genus Acomys represent the first example of full skin autotomy in mammals. Acomys cahirinus has evolved extremely weak skin as a strategy to avoid predation and is able to repeatedly regenerate healthy tissue without scar after severe skin injury or full-thickness ear punches. Extracellular matrix (ECM) composition is a critical regulator of wound repair and scar formation and previous studies have suggested that alterations in its expression may be responsible for the differences in regenerative capacity observed between Mus musculus and A. cahirinus , yet analysis of this critical tissue component has been limited in previous studies by its insolubility and resistance to extraction. Here, we utilize a 2-step ECM-optimized extraction to perform proteomic analysis of tissue composition during wound repair after full-thickness ear punches in A. cahirinus and M. musculus from weeks 1 to 4 post-injury. We observe changes in a wide range of ECM proteins which have been previously implicated in wound regeneration and scar formation, including collagens, coagulation and provisional matrix proteins, and matricryptic signaling peptides. We additionally report differences in crosslinking enzyme activity and ECM protein solubility between Mus and Acomys. Furthermore, we observed rapid and sustained increases in CD206, a marker of pro-regenerative M2 macrophages, in Acomys, whereas little or no increase in CD206 was detected in Mus. Together, these findings contribute to a comprehensive understanding of tissue cues which drive the regenerative capacity of Acomys and identify a number of potential targets for future pro-regenerative therapies.

Trans-Omics analysis of post injury thrombo-inflammation identifies endotypes and trajectories in trauma patients.

Understanding and managing the complexity of trauma-induced thrombo-inflammation necessitates an innovative, data-driven approach. This study leveraged a trans-omics analysis of longitudinal samples from trauma patients to illuminate molecular endotypes and trajectories that underpin patient outcomes, transcending traditional demographic and physiological characterizations. We hypothesize that trans-omics profiling reveals underlying clinical differences in severely injured patients that may present with similar clinical characteristics but ultimately have very different responses to treatment and clinical outcomes. Here we used proteomics and metabolomics to profile 759 of longitudinal plasma samples from 118 patients at 11 time points and 97 control subjects. Results were used to define distinct patient states through data reduction techniques. The patient groups were stratified based on their shock severity and injury severity score, revealing a spectrum of responses to trauma and treatment that are fundamentally tied to their unique underlying biology. Ensemble models were then employed, demonstrating the predictive power of these molecular signatures with area under the receiver operating curves of 80 to 94% for key outcomes such as INR, ICU-free days, ventilator-free days, acute lung injury, massive transfusion, and death. The molecularly defined endotypes and trajectories provide an unprecedented lens to understand and potentially guide trauma patient management, opening a path towards precision medicine. This strategy presents a transformative framework that aligns with our understanding that trauma patients, despite similar clinical presentations, might harbor vastly different biological responses and outcomes.

Blockade of Osteoclast-Mediated Bone Resorption With a RANKL-Inhibitor Enhances Bone Formation in a Rat Spinal Fusion Model.

STUDY DESIGN: Rat spine fusion model. OBJECTIVE: The present study aimed to determine whether administration of osteoprotegerin (OPG) in a rat model of spinal fusion increases bone volume, bone density, and decreases osteoclasts in the fusion mass. SUMMARY OF BACKGROUND DATA: OPG is a soluble RANK-ligand inhibitor that blocks osteoclast differentiation and activation. This makes it a potential agent to control the remodeling process and enhance bone mass during spinal fusion. MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats received a one-level spinal fusion of L4-L5 with bone allograft. Rats were then divided into four groups according to initiation of treatment: (1) saline on day 0 (saline), (2) OPG on day 0 (OPG D0), (3) OPG on day 10 (OPG D10), and (4) OPG on day 21 (OPG D21) postsurgery. After their initial injection, rats received weekly subcutaneous injections of OPG (10 mg/kg) and were euthanized six weeks postsurgery. MicroCT analysis of the fusion site and histological analysis of bone surface for quantification of osteoclast lining was performed. RESULTS: Increased bone volume in the fusion site and around the spinous process was seen in OPG D0 and OPG D10 when compared with saline. Mean trabecular thickness was greater in all groups receiving OPG compared with saline, with OPG D0 and OPG D10 having significantly greater mean trabecular thickness than OPG D21. All OPG groups had less bone surface lined with osteoclasts when compared with Saline, with OPG D0 and OPG D10 having fewer than OPG D21. CONCLUSIONS: This study indicates that OPG inhibited osteoclast bone resorption, which led to greater bone at the fusion site. Future studies investigating OPG on its own or in combination with an osteogenic factor to improve spinal fusion outcomes are warranted to further elucidate its potential therapeutic effect.

Analysis of Physeal Fractures from the United States National Trauma Data Bank.

BACKGROUND: Pediatric long-bone physeal fractures can lead to growth deformities. Previous studies have reported that physeal fractures make up 18-30% of total fractures. This study aimed to characterize physeal fractures with respect to sex, age, anatomic location, and Salter-Harris (SH) classification from a current multicenter national database. METHODS: A retrospective cohort study was performed using the 2016 United States National Trauma Data Bank (NTDB). Patients ≤ 18 years of age with a fracture of the humerus, radius, ulna, femur, tibia, or fibula were included. RESULTS: The NTDB captured 132,018 patients and 58,015 total fractures. Physeal fractures made up 5.7% (3291) of all long-bone fractures, with males accounting for 71.0% (2338). Lower extremity physeal injuries comprised 58.6% (1929) of all physeal fractures. The most common site of physeal injury was the tibia comprising 31.8% (1047), 73.9% (774) of which were distal tibia fractures. Physeal fractures were greatest at 11 years of age for females and 14 years of age for males. Most fractures were SH Type II fractures. DISCUSSION AND CONCLUSIONS: Our analysis indicates that 5.7% of pediatric long-bone fractures involved the physis, with the distal tibia being the most common. These findings suggest a lower incidence of physeal fractures than previous studies and warrant further investigation.

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

A timeseries analysis of the fracture callus extracellular matrix proteome during bone fracture healing.

While most bones fully self-heal, certain diseases require bone allograft to assist with fracture healing. Bone allografts offer promise as treatments for such fractures due to their osteogenic properties. However, current bone allografts made of decellularized bone extracellular matrix (ECM) have high failure rates, and thus grafts which improve fracture healing outcomes are needed. Understanding specific changes to the ECM proteome during normal fracture healing would enable the identification of key proteins that could be used enhance osteogenicity of bone allograft. Here, we performed a timeseries analysis of the fracture callus in mice to investigate proteomic and mineralization changes to the ECM at key stages of fracture healing. We found that changes to the ECM proteome largely coincide with the distinct phases of fracture healing. Basement membrane proteins (AGRN, COL4, LAMA), cartilage proteins (COL2A1, ACAN), and collagen crosslinking enzymes (LOXL, PLOD, ITIH) were initially upregulated, followed by bone specific proteoglycans and collagens (IBSP, COL1A1). Various tissue proteases (MMP2, 9, 13, 14; CTSK, CTSG, ELANE) were expressed at different levels throughout fracture healing. These changes coordinated with mineralization of the fracture callus, which increased steeply during the initial stages of healing. Interestingly the later timepoint was characterized by a response to wound healing and high expression of clotting factors (F2, 7, 9, 10). We identified ELANE and ITIH2 as tissue remodeling enzymes having no prior known involvement with fracture healing. This data can be further mined to identify regenerative proteins for enhanced bone graft design.

Anti-VEGF antibody delivered locally reduces bony bar formation following physeal injury in rats.

Physeal injuries can result in the formation of a "bony bar" which can lead to bone growth arrest and deformities in children. Vascular endothelial growth factor (VEGF) has been shown to play a role in bony bar formation, making it a potential target to inhibit bony repair tissue after physeal injury. The goal of this study was to investigate whether the local delivery of anti-VEGF antibody (α-VEGF; 7.5 µg) from alginate:chitosan hydrogels to the tibial physeal injury site in rats prevents bony bar formation. We tested the effects of quick or delayed delivery of α-VEGF using both 90:10 and 50:50 ratio alginate:chitosan hydrogels, respectively. Male and female 6-week-old Sprague-Dawley rats received a tibial physeal injury and the injured site injected with alginate-chitosan hydrogels: (1) 90:10 (Quick Release); (2) 90:10 + α-VEGF (Quick Release + α-VEGF); (3) 50:50 (Slow Release); (4) 50:50 + α-VEGF (Slow Release + α-VEGF); or (5) Untreated. At 2, 4, and 24 weeks postinjury, animals were euthanized and tibiae assessed for bony bar and vessel formation, repair tissue type, and limb lengthening. Our results indicate that Quick Release + α-VEGF reduced bony bar and vessel formation, while also increasing cartilage repair tissue. Further, the quick release of α-VEGF neither affected limb lengthening nor caused deleterious side-effects in the adjacent, uninjured physis. This α-VEGF treatment, which inhibits bony bar formation without interfering with normal bone elongation, could have positive implications for children suffering from physeal injuries.

In vivo degradation rate of alginate-chitosan hydrogels influences tissue repair following physeal injury.

The physis is a cartilaginous tissue in children's long bones that is responsible for bone elongation. Physeal injuries can heal with bony repair tissue known as a "bony bar," and this can cause growth deformities. Current treatments involve surgical resection of the bony bar and insertion of inert materials in hopes of preventing bony bar re-formation and preserving bone elongation. However, these materials frequently fail and the bony bar commonly returns. This study investigated alginate-chitosan hydrogels as interpositional materials to block bony bar formation in a rat model of physeal injury. Further, biomaterial properties such as substrate stiffness, permeability, and degradation rate were studied. Different ratio alginate:chitosan hydrogels with or without calcium cross-linking were tested for their inhibition of bony bar formation and restoration of the injured physis. Alginate:chitosan were mixed (a) 90:10 with calcium (90:10 + Ca); (b) 50:50 with calcium (50:50 + Ca); (c) 50:50 without calcium (50:50 - Ca); and (d) 50:50 made with irradiated alginate (IA) and without calcium. We found that repair tissue was determined primarily by the in vivo degradation rate of alginate-chitosan hydrogels. 90:10 + Ca had a slow degradation rate, prevented cellular infiltration, and produced the most bony bar tissue while having softer, more permeable material properties. IA had the fastest degradation, showed high cellular infiltration, and produced the most cartilage-like tissue while having stiffer, less permeable material properties. Our results suggest that the in vivo biomaterial degradation rate is a dynamic property that can be optimized to influence cell fate and tissue repair in physeal injuries.

Rabbit Model of Physeal Injury for the Evaluation of Regenerative Medicine Approaches.

Physeal injuries can lead to bony repair tissue formation, known as a bony bar. This can result in growth arrest or angular deformity, which is devastating for children who have not yet reached their full height. Current clinical treatment involves resecting the bony bar and replacing it with a fat graft to prevent further bone formation and growth disturbance, but these treatments frequently fail to do so and require additional interventions. Novel treatments that could prevent bone formation but also regenerate the injured physeal cartilage and restore normal bone elongation are warranted. To test the efficacy of these treatments, animal models that emulate human physeal injury are necessary. The rabbit model of physeal injury quickly establishes a bony bar, which can then be resected to test new treatments. Although numerous rabbit models have been reported, they vary in terms of size and location of the injury, tools used to create the injury, and methods to assess the repair tissue, making comparisons between studies difficult. The study presented here provides a detailed method to create a rabbit model of proximal tibia physeal injury using a two-stage procedure. The first procedure involves unilateral removal of 25% of the physis in a 6-week-old New Zealand white rabbit. This consistently leads to a bony bar, significant limb length discrepancy, and angular deformity within 3 weeks. The second surgical procedure involves bony bar resection and treatment. In this study, we tested the implantation of a fat graft and a photopolymerizable hydrogel as a proof of concept that injectable materials could be delivered into this type of injury. At 8 weeks post-treatment, we measured limb length, tibial angle, and performed imaging and histology of the repair tissue. By providing a detailed, easy to reproduce methodology to perform the physeal injury and test novel treatments after bony bar resection, comparisons between studies can be made and facilitate translation of promising therapies toward clinical use. Impact Statement This study provides details to create a rabbit model of physeal injury that can facilitate comparisons between studies and test novel regenerative medicine approaches. Furthermore, this model mimics the human, clinical situation that requires a bony bar resection followed by treatment. In addition, identification of a suitable treatment can be seen in the correction of the growth deformity, allowing this model to facilitate the development of novel physeal cartilage regenerative medicine approaches.

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies.

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

Estradiol-mediated improvements in adipose tissue insulin sensitivity are related to the balance of adipose tissue estrogen receptor α and ß in postmenopausal women.

We recently demonstrated that short-term estradiol (E2) treatment improved insulin-mediated suppression of lipolysis in postmenopausal women, but to a greater extent in those who were late compared to early postmenopausal. In this follow-up study we tested whether subcutaneous adipose tissue (SAT) expression of estrogen receptors (ER) α and ß differs between early and late postmenopausal women. We further tested whether the balance of ERα to ERß in SAT determined the effect of E2 on SAT insulin sensitivity. The present study included 35 women who were ≤6 years past menopause (EPM; n = 16) or ≥10 years past menopause (LPM; n = 19). Fasted SAT samples were taken following 1-week transdermal E2 treatment or placebo (PL) in a random cross-over design. Samples were analyzed for nuclear/cytosolic protein content and mRNA expression using Western blot and qPCR, respectively. While ESR1 increased slightly (~1.4-fold) following E2 treatment in both groups, ERα and ERß protein expression did not differ between groups at baseline or in response to E2. However, the balance of ERα/ERß protein in the SAT nuclear fraction increased 10% in EPM compared to a 25% decrease in LPM women (group x treatment interaction, p<0.05). A greater proportion of ERα/ERß protein in the nuclear fraction of SAT at baseline (placebo day) was associated with greater reduction in SAT insulin resistance (i.e., better suppression of lipolysis, EC50) in response to E2 (r = -0.431, p<0.05). In conclusion, there do not appear to be differences in the proportion of adipose tissue ERα/ERß protein in late, compared to early, postmenopausal women. However, the balance of ERα/ERß may be important for E2-mediated improvement in adipose tissue insulin sensitivity. TRIAL REGISTRATION: Clinical Trials#: NCT01605071.

Time since menopause and skeletal muscle estrogen receptors, PGC-1α, and AMPK.

OBJECTIVE: Short-term administration of estradiol (E2) improves insulin-stimulated glucose disposal rate in early postmenopausal (EPM) women compared with a reduction in late postmenopausal (LPM) women. The underlying mechanisms by which E2 action on glucose disposal rate reversed from beneficial early to harmful late in menopause is unknown, but might include adverse changes in estrogen receptors (ERs) or other biomarkers of cellular energy metabolism with age or duration of estrogen deficiency. METHODS: We retrospectively analyzed skeletal muscle samples from 27 postmenopausal women who were 6 years or less past menopause (EPM; n = 13) or at least 10 years past menopause (LPM; n = 14). Fasted skeletal muscle (vastus lateralis) samples were collected after 1 week administration of transdermal E2 or placebo, in random cross-over design. RESULTS: Compared with EPM, LPM had reduced skeletal muscle ERα and ERß nuclear protein. Short-term E2 treatment did not change nuclear ERα or ERß, but decreased cytosolic ERα, so the proportion of ERα in the nucleus compared with the cytosol tended to increase. There was a group-by-treatment interaction (P < 0.05) for nuclear proliferator-activated receptor γ co-activator 1-α and phosphorylated adenosine monophosphate-activated protein kinase, such that E2 increased these proteins in EPM, but decreased these proteins in LPM. CONCLUSIONS: These preliminary studies of skeletal muscle from early and late postmenopausal women treated with E2 suggest there may be declines in skeletal muscle ER and changes in the E2-mediated regulation of cellular energy homeostasis with increasing time since menopause.

Region-specific effects of oestradiol on adipose-derived stem cell differentiation in post-menopausal women.

The goal of this study was to determine the effect of acute transdermal 17ß-oestradiol (E2 ) on the adipogenic potential of subcutaneous adipose-derived stem cells (ASC) in post-menopausal women. Post-menopausal women (n = 11; mean age 57 ± 4.5 years) were treated for 2 weeks, in a randomized, cross-over design, with transdermal E2 (0.15 mg) or placebo patches. Biopsies of abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) were obtained after each treatment and mature adipocytes were analysed for cell size and ASC for their capacity for proliferation (growth rate), differentiation (triglyceride accumulation) and susceptibility to tumour necrosis factor alpha-induced apoptosis. Gene expression of oestrogen receptors α and ß (ESR1 and ESR2), perilipin 1 and hormone-sensitive lipase (HSL), was also assessed. In FEM SAT, but not AB SAT, 2 weeks of E2 significantly (P = 0.03) increased ASC differentiation and whole SAT HSL mRNA expression (P = 0.03) compared to placebo. These changes were not associated with mRNA expression of oestrogen receptors α and ß, but HSL expression was significantly increased in FEM SAT with transdermal E2 treatment. Adipose-derived stem cells proliferation and apoptosis did not change in either SAT depot after E2 compared with placebo. Short-term E2 appeared to increase the adipogenic potential of FEM, but not AB, SAT in post-menopausal women with possible implications for metabolic disease. Future studies are needed to determine longer term impact of E2 on regional SAT accumulation in the context of positive energy imbalance.

Femoral lipectomy increases postprandial lipemia in women.

Femoral subcutaneous adipose tissue (SAT) appears to be cardioprotective compared with abdominal SAT, possibly through better triglyceride (TG) sequestration. We hypothesized that removal of femoral SAT would increase postprandial TG through a reduction in dietary fatty acid (FA) storage. Normal-weight (means ± SD; BMI 23.9 ± 2.6 kg/m(2)) women (n = 29; age 45 ± 6 yr) were randomized to femoral lipectomy (LIPO) or control (CON) and followed for 1 yr. Regional adiposity was measured by DEXA and CT. A liquid meal labeled with [(14)C]oleic acid was used to trace the appearance of dietary FA in plasma (6-h postprandial TG), breath (24-h oxidation), and SAT (24-h [(14)C]TG storage). Fasting LPL activity was measured in abdominal and femoral SAT. DEXA leg fat mass was reduced after LIPO vs. CON (Δ-1.4 ± 0.7 vs. 0.1 ± 0.5 kg, P < 0.001) and remained reduced at 1 yr (-1.1 ± 1.4 vs. -0.2 ± 0.5 kg, P < 0.05), as did CT thigh subcutaneous fat area (-39.6 ± 36.6 vs. 4.7 ± 14.6 cm(2), P < 0.05); DEXA trunk fat mass and CT visceral fat area were unchanged. Postprandial TG increased (5.9 ± 7.7 vs. -0.6 ± 5.3 × 10(3) mg/dl, P < 0.05) and femoral SAT LPL activity decreased (-21.9 ± 22.3 vs. 10.5 ± 26.5 nmol·min(-1)·g(-1), P < 0.05) 1 yr following LIPO vs. CON. There were no group differences in (14)C-labeled TG appearing in abdominal and femoral SAT or elsewhere. In conclusion, femoral fat remained reduced 1 yr following lipectomy and was accompanied by increased postprandial TG and reduced femoral SAT LPL activity. There were no changes in storage of meal-derived FA or visceral fat. Our data support a protective role for femoral adiposity on circulating TG independent of dietary FA storage and visceral adiposity.

Postprandial triglycerides and adipose tissue storage of dietary fatty acids: impact of menopause and estradiol.

OBJECTIVES: Postprandial lipemia worsens after menopause, but the mechanism remains unknown. The hypothesized menopause-related postprandial lipemia would be (1) associated with reduced storage of dietary fatty acids (FA) as triglyceride (TG) in subcutaneous adipose tissue (SAT) and (2) improved by short-term estradiol (E2 ). METHODS: Twenty-three pre- (mean ± SD: 42 ± 4 years) and 22 postmenopausal (55 ± 4 years) women with similar total adiposity were studied. A subset of postmenopausal women (n = 12) were studied following 2 weeks of E2 (0.15 mg) and matching placebo in a random, cross-over design. A liquid meal containing (14) C-oleic acid traced appearance of dietary FA in: serum (postprandial TG), breath (oxidation), and abdominal and femoral SAT (TG storage). RESULTS: Compared to premenopausal women, healthy, lean, postmenopausal women had increased postprandial glucose and insulin and trend for higher TG but had similar dietary FA oxidation and storage. Adipocytes were larger in post- compared to premenopausal women, particularly in femoral SAT. Short-term E2 reduced postprandial TG and insulin but had no effect on oxidation or storage of dietary FA. E2 increased the proportion of small adipocytes in femoral (but not abdominal) SAT. CONCLUSIONS: Short-term E2 attenuated menopause-related increases in postprandial TG and increased femoral adipocyte hyperplasia but not through increased net storage of dietary FA.

Neisserial pilin genes display extensive interspecies diversity.

All Neisseria live in association with host cells, however, little is known about the genetic potential of nonpathogenic Neisseria species to express attachment factors such as pili. In this study, we demonstrate that type IV pilin-encoding genes are present in a wide range of Neisseria species. N. sicca, N. subflava, and N. elongata each contain two putative pilE genes arranged in tandem, while single genes were identified in N. polysaccharea, N. mucosa, and N. denitrificans. Neisserial pilE genes are highly diverse and display features consistent with a history of horizontal gene transfer.